Combination Immunotherapy Compositions Against Cancer and Methods

ABSTRACT

Disclosed are immunotherapeutic compositions and the concurrent use of combinations of such compositions for the improved induction of therapeutic immune responses and/or for the prevention, amelioration and/or treatment of disease, including, but not limited to, cancer and infectious disease.

GOVERNMENT RIGHTS

This invention was created in the performance of a Cooperative Researchand Development Agreement with the National Institutes of Health, anAgency of the Department of Health and Human Services. The Government ofthe United States has certain rights in this invention.

STATEMENT REGARDING JOINT RESEARCH AGREEMENT

This invention was made by or on behalf of parties to a CooperativeResearch and Development Agreement, executed May 8, 2008. The parties tothe Cooperative Research and Development Agreement are: GlobeImmune,Inc. and the U.S. Department of Health and Human Services, asrepresented by National Cancer Institute, an Institute, Center orDivision of the National Institutes of Health.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing submitted electronically asa text file by EFS-Web. The text file, named “3923-24-PCT ST25”, has asize in bytes of 47 KB, and was recorded on 16 Apr. 2010. Theinformation contained in the text file is incorporated herein byreference in its entirety pursuant to 37 CFR § 1.52(e)(5).

FIELD OF THE INVENTION

The present invention generally relates to the concurrent use of twodifferent immunotherapeutic compositions for the improved induction oftherapeutic immune responses and/or for the prevention, ameliorationand/or treatment of disease, including, but not limited to, cancer andinfectious disease.

BACKGROUND OF THE INVENTION

Immunotherapeutic compositions, including vaccines, are one of the mostcost-effective measures available to the health care industry for theprevention and treatment of disease. There remains, however, an urgentneed to develop safe and effective immunotherapy strategies andadjuvants for a variety of diseases, including those caused by orassociated with infection by pathogenic agents, cancers, genetic defectsand other disorders of the immune system. For the treatment of cancerand many infectious diseases, including viral diseases and diseasescaused by intracellular pathogens, it is desirable to provideimmunotherapy that elicits a cell-mediated (cellular) immune response,although many vaccines are directed primarily or entirely to elicitationof humoral immunity. Indeed, a disadvantage of many subunit vaccines, aswell as many killed or attenuated pathogen vaccines, is that while theyappear to stimulate a strong humoral immune response, they fail toelicit protective cell-mediated immunity.

Cancer is a leading cause of death worldwide, and the development ofeffective therapies for cancer continues to be one of the most activeareas of research. Although a variety of innovative approaches to treatand prevent cancers have been proposed, many cancers continue to have ahigh rate of mortality and may be difficult to treat or relativelyunresponsive to conventional therapies. Novel discoveries in cancerbiology have provided the opportunity to design target-specificanti-cancer agents and have fostered advances in drug and immunotherapydevelopment. These discoveries make it possible to design molecules andtherapeutic compositions with high selectivity against specific targetsin cancer cells.

Numerous immunotherapy studies have been reported comparing vaccineplatforms that target the same antigen, in terms of their ability toinduce immune cell activity and antitumor effects (e.g., see Weide etal., Immunol Lett 2008 Jan. 15; 115(1):33-42; Riezebos-Brilman et al.,Gene Ther 2007 December; 14(24):1695-704; Naslund et al., J Immunol 2007Jun. 1; 178(11):6761-9; Mylin et al., J Virol 2000 August;74(15):6922-34; Millar et al., Cell Immunol 2007 November-December;250(1-2):55-67; Hodge et al., Cancer Res 2003 Nov. 15; 63(22):7942-9;Chan et al., Gene Ther 2006 October; 13(19):1391-402; Casimiro et al., JVirol 2003 June; 77(11):6305-13; and Bos et al., J Immunol 2007 Nov. 1;179(9):6115-22). Millar et al. showed that the functionality of T-cellpopulations induced by two different vectors (rV and recombinantadenovirus) targeting the same antigen did not differ (Millar et al.,Cell Immunol 2007 November-December; 250(1-2):55-67).

The antitumor efficacy of the diversified prime and boost vaccineregimen of recombinant vaccinia (rV) and recombinant fowlpox (rF)viruses containing murine B7-1, ICAM-1, and LFA-3 genes as well as thehuman carcinoembryonic antigen (CEA) gene (rV/F-CEA/TRICOM) haspreviously been reported in preclinical models (Hodge et al., Cancer Res2003 Nov. 15; 63(22):7942-9; Hodge et al., Cancer Res 1999 Nov. 15;59(22):5800-7; Hodge et al., Clin Cancer Res 2003 May; 9(5):1837-49;Grosenbach et al., Cancer Res 2001 Jun. 1; 61(11):4497-505; Greiner etal., Cancer Res 2002 Dec. 1; 62(23):6944-51; Arlen et al., Crit RevImmunol 2007; 27(5):451-62). Recently, the antitumor effects of arecombinant Saccharomyces cerevisiae (yeast-CEA) vaccine were alsodocumented in preclinical models (Bernstein et al., Vaccine 2008 Jan.24; 26(4):509-21; Wansley et al., Clin Cancer Res 2008 Jul. 1;14(13):4316-25). The induction of immune response after vaccination witheither rV/FCEA/TRICOM or yeast-CEA has been documented, and theantitumor effects elicited by either vaccine are mainly attributed tothe induction of CEA-specific T-cell populations.

Several studies have documented that the induction of a more diverseT-cell population is advantageous in mounting an immune response invarious models of disease, including cancer (Dudley et al., Cancer J2000 March-April; 6(2):69-77; Dutoit et al., Cancer Res 2001 Aug. 1;61(15):5850-6; Echchakir et al., Int Immunol 2000 April; 12(4):537-46;Ferradini et al., Cancer Res 1992 Sep. 1; 52(17):4649-54; Messaoudi etal., Science 2002 Nov. 29; 298(5599):1797-800; Nikolich-Zugich et al.,Nat Rev Immunol 2004 February; 4(2):123-32; Sportes et al., J Exp Med2008 Jul. 7; 205(7):1701-14; Zhou et al., Cancer Res 2005 Feb. 1;65(3):1079-88). However, there are no reports of concurrent use ofvaccines that target the same antigen. Following studies targeting thesame antigen, such as those described above, investigators historicallyeither choose the most efficacious vaccine for further study, or employa diversified prime and boost strategy to amplify the T-cell response.For example, a diversified prime-boost vaccination strategy withrecombinant vaccinia and fowlpox vectors targeting CEA (Hodge et al.,Vaccine 1997 April-May; 15(6-7):759-68; Marshall et al., J Clin Oncol2000 Dec. 1; 18(23):3964-73), was employed because the immune responseto the first vaccine has been shown to reduce the effects of subsequentvaccinations with the same vector (Naslund et al., 2007, supra;Grosenbach et al., 2001, supra, Hodge et al., 1997, supra, and Wu etal., J Virol 2005 July; 79(13):8024-31.). Similar results demonstratingthe clear advantages of a diversified prime-boost strategy have beendescribed in a variety of cancer and other disease models, including HIVand malaria (Wu et al, 2005, supra; Pancholi et al., J Infect Dis 2000July; 182(1):18-27; Barnett et al., AIDS Res Hum Retroviruses 1998October; 14 Suppl 3:S299-309; Dunachie et al., J Exp Biol 2003 November;206(Pt 21):3771-9; McMichael, Annu Rev Immunol 2006; 24:227-55; Moore etal., Immunol Rev 2004 June; 199:126-43). The enhanced responses observedin these studies have been attributed to amplification of the relevantpopulation of antigen-specific T-cells, but again, a diversifiedprime-boost approach was used to achieve these results.

Accordingly, despite advances in cancer therapy and infectious diseaseimmunotherapy/vaccine technology, there remains an urgent need toimprove safe and effective immunotherapy approaches to the treatmentsuch diseases.

SUMMARY OF THE INVENTION

One embodiment of the invention relates to a method to prevent,ameliorate or treat at least one symptom of a cancer in an individual,to increase survival of an individual with cancer, and/or to induce atherapeutic immune response against one or more cancer antigens in theindividual. The method includes the step of administering to theindividual: (a) a first immunotherapy composition comprising arecombinant vaccinia virus comprising nucleic acid sequences encoding atleast one costimulatory molecule and nucleic acid sequences encoding atleast one cancer antigen or immunogenic domain thereof; and (b) a secondimmunotherapy composition comprising a yeast vehicle comprising at leastone cancer antigen or immunogenic domain thereof. The first and secondimmunotherapy compositions are administered concurrently to theindividual. In one aspect, the recombinant vaccinia virus comprisesnucleic acid sequences encoding costimulatory molecules B7-1, ICAM-1 andLFA-3.

Another embodiment of the invention relates to a method to prevent,ameliorate or treat at least one symptom of a cancer in an individual,to increase survival of an individual with cancer, and/or to induce atherapeutic immune response against one or more cancer antigens in theindividual. The method includes a step of administering to theindividual: (a) a first immunotherapy composition comprising: (i) arecombinant vaccinia virus comprising nucleic acid sequences encodingB7-1, ICAM-1 and LFA-3 or biologically active portions thereof, and anucleic acid sequence encoding at least one cancer antigen orimmunogenic domain thereof; and (ii) a recombinant fowlpox viruscomprising a nucleic acid sequence encoding GM-CSF or a biologicallyactive portion thereof; and (b) a second immunotherapy compositioncomprising a yeast vehicle comprising at least one cancer antigen orimmunogenic domain thereof. The first and second immunotherapycompositions are administered concurrently to the individual. In oneaspect of this embodiment, at least one week after the firstadministration, the method additionally includes the step ofadministering to the individual: (a) a third immunotherapy compositioncomprising: (i) a recombinant fowlpox virus comprising nucleic acidsequences encoding B7-1, ICAM-1 and LFA-1 or biologically activeportions thereof, and a nucleic acid sequence encoding the at least onecancer antigen or immunogenic domain thereof; and (ii) a recombinantfowlpox virus comprising a nucleic acid sequence encoding GM-CSF or abiologically active portion thereof; and (b) the second immunotherapycomposition comprising a yeast vehicle comprising the at least onecancer antigen or immunogenic domain thereof. The second and thirdimmunotherapy compositions are administered concurrently to theindividual.

Yet another embodiment of the invention relates to the use of acombination of immunotherapy compositions in the preparation ofmedicaments for concurrent administration to an individual to prevent,ameliorate or treat at least one symptom of cancer in the individual, orto increase survival of an individual who has cancer, and/or to induce atherapeutic immune response against one or more antigens in theindividual. The immunotherapy compositions comprise: (a) a firstimmunotherapy composition comprising: (i) a recombinant vaccinia viruscomprising nucleic acid sequences encoding B7-1, ICAM-1 and LFA-3 orbiologically active portions thereof, and nucleic acid sequencesencoding at least one cancer antigen or immunogenic domain thereof; and(ii) a recombinant fowlpox virus comprising a nucleic acid sequenceencoding GM-CSF or a biologically active portion thereof; and (b) asecond immunotherapy composition comprising a yeast vehicle comprisingat least one cancer antigen or immunogenic domain thereof. In oneaspect, the immunotherapy compositions further comprise a thirdimmunotherapy composition comprising: (i) a recombinant fowlpox viruscomprising nucleic acid sequences encoding B7-1, ICAM-1 and LFA-3 orbiologically active portions thereof, and nucleic acid sequencesencoding the at least one cancer antigen or immunogenic domain thereof;and (ii) a recombinant fowlpox virus comprising a nucleic acid sequenceencoding GM-CSF or a biologically active portion thereof.

One embodiment of the invention relates to a method to prevent,ameliorate or treat at least one symptom of a disease or condition in anindividual. The method includes a step of administering to theindividual: (a) a first immunotherapy composition comprising arecombinant virus comprising the virus genome or portions thereof; and(b) a second immunotherapy composition comprising a yeast vehicle. Thefirst and second immunotherapy compositions are administeredconcurrently to the individual.

Another embodiment of the invention relates to a method to induce atherapeutic immune response against one or more antigens in anindividual. The method includes the step of administering to theindividual: (a) a first immunotherapy composition comprising arecombinant virus comprising the virus genome or portions thereof; and(b) a second immunotherapy composition comprising a yeast vehicle. Thefirst and second immunotherapy compositions are administeredconcurrently to the individual.

Another embodiment of the invention relates to a method to increasesurvival of an individual who has a disease or condition. The methodincludes a step of administering to the individual: (a) a firstimmunotherapy composition comprising a recombinant virus comprising thevirus genome or portions thereof; and (b) a second immunotherapycomposition comprising a yeast vehicle. The first and secondimmunotherapy compositions are administered concurrently to theindividual.

Yet another embodiment of the invention relates to the use of acombination of immunotherapy compositions in the preparation ofmedicaments for concurrent administration to an individual to prevent,ameliorate or treat at least one symptom of a disease or condition inthe individual. The immunotherapy compositions include: (a) a firstimmunotherapy composition comprising a recombinant virus comprising thevirus genome or portions thereof; and (b) a second immunotherapycomposition comprising a yeast vehicle. The compositions are formulatedfor concurrent administration.

Another embodiment of the invention relates to the use of a combinationof immunotherapy compositions in the preparation of medicaments forconcurrent administration to an individual to induce a therapeuticimmune response against one or more antigens in the individual. Theimmunotherapy compositions comprise: (a) a first immunotherapycomposition comprising a recombinant virus comprising the virus genomeor portions thereof; and (b) a second immunotherapy compositioncomprising a yeast vehicle. One or both of the first and secondimmunotherapy compositions comprises at least one antigen or immunogenicdomain thereof. The compositions are formulated for concurrentadministration.

Yet another embodiment of the invention relates to the use of acombination of immunotherapy compositions in the preparation ofmedicaments for concurrent administration to an individual who has adisease or condition to increase survival of the individual. Theimmunotherapy compositions comprise: (a) a first immunotherapycomposition comprising a recombinant virus comprising the virus genomeor portions thereof; and (b) a second immunotherapy compositioncomprising a yeast vehicle. One or both of the first and secondimmunotherapy compositions comprises at least one antigen or immunogenicdomain thereof. The compositions are formulated for concurrentadministration.

Yet another embodiment of the invention relates to a compositioncomprising: (a) a first immunotherapy composition comprising arecombinant virus comprising the virus genome or portions thereof; and(b) a second immunotherapy composition comprising a yeast vehicle. Oneor both of the first and second immunotherapy compositions comprises atleast one antigen or immunogenic domain thereof. The first and secondimmunotherapy compositions are provided in admixture.

Another embodiment of the invention relates to a kit comprising thefollowing immunotherapy compositions: (a) a first immunotherapycomposition comprising a recombinant vaccinia virus comprising nucleicacid sequences encoding at least one costimulatory molecule and nucleicacid sequences encoding at least one antigen or immunogenic domainthereof and (b) a second immunotherapy composition comprising a yeastvehicle comprising at least one antigen or immunogenic domain thereof.In one aspect, the recombinant vaccinia virus comprises nucleic acidsequences encoding costimulatory molecules B7-1, ICAM-1 and LFA-3. Inone aspect, the antigen is a cancer antigen. In one aspect, the antigenis a modified CEA comprising a CAP-1-6D epitope. Other aspects of thekit of the invention are described below.

Another embodiment of the invention relates to a kit comprising thefollowing immunotherapy compositions: (a) a first immunotherapycomposition comprising: (i) a recombinant vaccinia virus comprisingnucleic acid sequences encoding B7-1, ICAM-1 and LFA-3 or biologicallyactive portions thereof, and a nucleic acid sequence encoding at leastone antigen or immunogenic domain thereof; and (ii) a recombinantfowlpox virus comprising a nucleic acid sequence encoding GM-CSF or abiologically active portion thereof; and (b) a second immunotherapycomposition comprising a yeast vehicle comprising at least one antigenor immunogenic domain thereof. In one aspect, the kit further comprisesa third immunotherapy composition comprising: (i) a recombinant fowlpoxvirus comprising nucleic acid sequences encoding B7-1, ICAM-1 and LFA-3or biologically active portions thereof, and a nucleic acid sequenceencoding the at least one antigen or immunogenic domain thereof; and(ii) a recombinant fowlpox virus comprising a nucleic acid sequenceencoding GM-CSF or a biologically active portion thereof. In one aspect,the antigen is a cancer antigen. In one aspect, the antigen is amodified CEA comprising a CAP-1-6D epitope. Other aspects of the kit ofthe invention are described below.

In any of the embodiments described herein (above or below), includingany of the embodiments related to a method, use, composition or kit ofthe invention, one or both of the first and second immunotherapycompositions (and/or third immunotherapy composition, in particularembodiments) comprises at least one antigen or immunogenic domainthereof. In one aspect, both the first and second immunotherapycompositions (and/or third immunotherapy composition, in particularembodiments) comprise at least one antigen or immunogenic domainthereof. In one aspect, the first immunotherapy composition comprises atleast one antigen or immunogenic domain thereof, and the second doesnot. In one aspect, the second immunotherapy composition comprises atleast one antigen or immunogenic domain thereof, and the first does not.In one aspect, each of the first and second immunotherapy compositionscomprises the same antigen or immunogenic domain thereof. In one aspect,the first and second immunotherapy compositions (and/or thirdimmunotherapy composition, in particular embodiments) comprise differentantigens or immunogenic domains thereof.

In one aspect of any of the embodiments or aspects of the inventiondescribed herein (above or below), including any of the embodimentsrelated to a method, use, composition or kit of the invention, the firstand/or third immunotherapy composition comprises a recombinant viruscomprising one or more nucleic acid sequences encoding one or moreimmunostimulatory molecules. In one aspect, the first and/or thirdimmunotherapy composition comprises a recombinant virus comprising thevirus genome or portions thereof and a nucleic acid sequence encoding atleast one antigen or immunogenic domain thereof, and a recombinant viruscomprising one or more nucleic acid sequences encoding one or moreimmunostimulatory molecules. In one aspect, the first and/or thirdimmunotherapy composition comprises a recombinant virus comprising anucleic acid sequence encoding the at least one antigen or immunogenicdomain thereof and one or more nucleic acid sequences encoding one ormore immunostimulatory molecules.

In one aspect of any of the embodiments or aspects of the inventiondescribed herein (above or below), including any of the embodimentsrelated to a method, use, composition or kit of the invention, therecombinant virus or viruses in the first and/or third immunotherapycomposition is a poxvirus. In one aspect, the recombinant virus orviruses in the first and/or third immunotherapy composition is arecombinant vaccinia virus. In one aspect, the vaccinia virus ismodified vaccinia Ankara (MVA). In one aspect, the recombinant virus orviruses in the first and/or third immunotherapy composition is a fowlpoxvirus.

In one aspect of any of the embodiments or aspects of the inventiondescribed herein (above or below), including any of the embodimentsrelated to a method, use, composition or kit of the invention, theimmunostimulatory molecules comprise one or more costimulatorymolecules. In one aspect, the immunostimulatory molecules include, butare not limited to, B7.1 (B7-1), B7.2 (B7-2), ICAM-1, LFA-3, 4-1BBL,CD59, CD40, CD40L and/or CD70. In one aspect, the immunostimulatorymolecules comprise one, two or all three of B7-1, ICAM-1, and LFA-3. Inone aspect, the immunostimulatory molecules comprise one or morecytokines, including, but not limited to, tumor necrosis factor-α(TNF-α), interleukin-6 (IL-6), granulocyte-macrophage-colony stimulatingfactor (GM-CSF), interferon-γ (IFN-γ), IFN-α, IFN-λ, interleukin-12(IL-12), RANTES, and interleukin-2 (IL-2). In one aspect, the cytokineis granulocyte-macrophage colony-stimulating factor (GM-CSF).

In one aspect of any of the embodiments or aspects of the inventiondescribed herein (above or below), including any of the embodimentsrelated to a method, use, composition or kit of the invention, thesecond immunotherapy composition comprises a yeast vehicle thatexpresses at least one antigen or immunogenic domain thereof. In oneaspect, the yeast vehicle in the second immunotherapy composition is awhole yeast. In one aspect, the yeast vehicle in the secondimmunotherapy composition is a whole, heat-killed yeast. In one aspect,the yeast vehicle in the second immunotherapy composition is fromSaccharomyces.

In one aspect of any of the method or use embodiments or aspects of theinvention described herein (above or below), the first and secondimmunotherapy compositions (and/or third immunotherapy composition incertain embodiments) are administered to different sites in theindividual. In another aspect, the first and second (and/or third)immunotherapy compositions are administered to the same site or toadjacent sites in the individual.

In another aspect of any of the method or use embodiments or aspects ofthe invention described herein (above or below), the method or usefurther comprises a step of boosting the individual with one or both (orall three, in certain embodiments) of the immunotherapy compositions. Inone aspect, the individual is boosted with the second immunotherapycomposition more frequently than with the first immunotherapycomposition.

In another aspect of any of the method or use embodiments or aspects ofthe invention described herein (above or below), wherein there is afirst and second immunotherapy composition, the method or use furthercomprises a step of boosting the individual with a third immunotherapycomposition comprising a recombinant virus comprising the virus genomeor portions thereof that is different from the first immunotherapycomposition. For example, in one aspect, the first immunotherapycomposition comprises a recombinant vaccinia virus, and the thirdimmunotherapy composition comprises a fowlpox virus.

In one aspect of any of the embodiments or aspects of the inventiondescribed herein, including any of the embodiments related to a method,use, composition or kit of the invention, either the first or the secondimmunotherapy composition is administered more frequently than theother. For example, in one aspect, when the first immunotherapycomposition is a virus-based immunotherapy composition, and the secondimmunotherapy composition is a yeast-based immunotherapy composition,the second immunotherapy composition may be administered more frequentlythan the first immunotherapy composition. For example, in one aspect, inbetween concurrent administrations of the first and second immunotherapycomposition, the second immunotherapy composition may be administeredone, two, three or more additional times.

In another aspect of any of the method or use embodiments or aspects ofthe invention described herein (above or below), the method furthercomprises a step of boosting the individual with another source of theantigen or immunogenic domain thereof.

In another aspect of any of the method or use embodiments or aspects ofthe invention described herein (above or below), the method furthercomprises a step of administering at least one biological responsemodifier to the individual.

In one aspect of any of the embodiments or aspects of the inventiondescribed herein (above or below), including any of the embodimentsrelated to a method, use, composition or kit of the invention, theindividual has cancer. In one aspect, the method or use reduces tumorburden or inhibits tumor growth in the individual. In one aspect of anyembodiment of the invention, including any of the embodiments related toa method, use, composition or kit of the invention, the antigen is froma cancer selected from the group of: melanomas, squamous cell carcinoma,breast cancers, head and neck carcinomas, thyroid carcinomas, softtissue sarcomas, bone sarcomas, testicular cancers, prostatic cancers,ovarian cancers, bladder cancers, skin cancers, brain cancers,angiosarcomas, hemangiosarcomas, mast cell tumors, leukemias, lymphomas,primary hepatic cancers, lung cancers, pancreatic cancers,gastrointestinal cancers, renal cell carcinomas, hematopoieticneoplasias or metastatic cancers thereof. In one aspect, the antigen isselected from the group of: carcinoembryonic antigen (CEA), pointmutated Ras oncoprotein, Brachyury, MUC-1, EGFR, BCR-Ab1, MART-1,MAGE-1, MAGE-3, GAGE, GP-100, MUC-2, normal and point mutated p53oncoproteins, PSMA, tyrosinase, TRP-1 (gp75), NY-ESO-1, TRP-2, TAG72,KSA, CA-125, PSA, HER-2/neu/c-erb/B2, hTERT, p′73, B-RAF, adenomatouspolyposis coli (APC), Myc, von Hippel-Lindau protein (VHL), Rb-1, Rb-2,androgen receptor (AR), Smad4, MDR1, Flt-3, BRCA-1, BRCA-2, pax3-fkhr,ews-fli-1, HERV-H, HERV-K, TWIST, Mesothelin, NGEP, modifications ofsuch antigens, splice variants of such antigens, and epitope agonists ofsuch antigens, as well as combinations of such antigens, and/orimmunogenic domains thereof, modifications thereof, variants thereof,and/or epitope agonists thereof. In one aspect, the antigen iscarcinoembryonic antigen (CEA), which in one embodiment, comprises aCAP1-6D epitope. In one aspect, the antigen is a modified CEA comprisinga CAP-1-6D epitope. In one aspect, the CEA comprises an amino acidsequence of SEQ ID NO:2 (encoded by a nucleic acid sequence representedherein as SEQ ID NO:1). In another aspect, the antigen is mutated Ras.In one aspect, the antigen is a multi-domain fusion protein, comprisingone or more fragments of Ras, each fragment comprising one or moremutations at position 12, 13, 59, 61 and/or 76 of Ras. In one aspect,the Ras fusion protein has an amino acid sequence of SEQ ID NO:4(encoded by a nucleic acid sequence represented herein as SEQ ID NO:3),SEQ ID NO:6 (encoded by a nucleic acid sequence represented herein asSEQ ID NO:5), SEQ ID NO:8 (encoded by a nucleic acid sequencerepresented herein as SEQ ID NO:7), and/or SEQ ID NO:10 (encoded by anucleic acid sequence represented herein as SEQ ID NO:9). In one aspect,the antigen is Brachyury. In one aspect, the antigen is MUC-1. In oneaspect, the antigen is EGFR. In one aspect, the antigen is BCR-Ab1.

In one aspect of any of the method or use embodiments or aspects of theinvention described herein (above or below), the method or use furtherincludes a step of treating the individual with chemotherapy, and/ortreating the individual with radiation therapy.

In another aspect of any of the embodiments or aspects of the inventiondescribed herein (above or below), including any of the embodimentsrelated to a method, use, composition or kit of the invention, theindividual has a disease caused by or associated with a pathogen. In oneaspect, the method or use reduces or prevents infection of theindividual by the pathogen. In one aspect, the method or use reducespathogen titer in the individual.

In one aspect of any embodiment of the invention, including any of theembodiments related to a method, use, composition or kit of theinvention, the antigen is selected from the group of: viral antigens,fungal antigens, bacterial antigens, helminth antigens, parasiticantigens, ectoparasite antigens, and protozoan antigens. In one aspect,the antigen is from a virus selected from: adenoviruses, arena viruses,bunyaviruses, coronaviruses, coxsackie viruses, cytomegaloviruses,Epstein-Barr viruses, flaviviruses, hepadnaviruses, hepatitis viruses,herpes viruses, influenza viruses, lentiviruses, measles viruses, mumpsviruses, myxoviruses, oncogenic viruses, orthomyxoviruses, papillomaviruses, papovaviruses, parainfluenza viruses, paramyxoviruses,parvoviruses, picornaviruses, pox viruses, rabies viruses, respiratorysyncytial viruses, reoviruses, rhabdoviruses, rubella viruses,togaviruses, varicella viruses, and T-lymphotrophic viruses. In oneaspect, the antigen is from an infectious agent from a genus selectedfrom the group consisting of: Aspergillus, Bordatella, Brugia, Candida,Chlamydia, Coccidia, Cryptococcus, Dirofilaria, Escherichia,Francisella, Gonococcus, Histoplasma, Leishmania, Mycobacterium,Mycoplasma, Paramecium, Pertussis, Plasmodium, Pneumococcus,Pneumocystis, Rickettsia, Salmonella, Shigella, Staphylococcus,Streptococcus, Toxoplasma, Vibriocholerae, and Yersinia. In one aspect,the antigen is from a bacterium from a genus selected from: Pseudomonas,Bordetella, Mycobacterium, Vibrio, Bacillus, Salmonella, Francisella,Staphylococcus, Streptococcus, Escherichia, Enterococcus, Pasteurella,and Yersinia.

BRIEF DESCRIPTION OF THE FIGURES OF THE INVENTION

FIGS. 1A-1I are graphs showing that vaccination with rV-CEA/TRICOM oryeast-CEA induces differential serum cytokine profiles (FIG. 1A=MIP1α,FIG. 1B=RANTES, FIG. 1C=GM-CSF, FIG. 1D=IL-6, FIG. 1E=IL-12p70, FIG.1F=IL-13, FIG. 1G=IL-1α, FIG. 1H=IL-1β and FIG. 11=IL-5.

FIGS. 2A-2F show that distinct TCR repertoires are induced fromvaccination with rV/F-CEA/TRICOM or yeast-CEA (Vα profiles are shown forno treatment (FIG. 2A), rV/F-CEA/TRICOM (FIG. 2B), and yeast-CEA (FIG.2C), and Vβ profiles are shown for no treatment (FIG. 2D),rV/F-CEA/TRICOM (FIG. 2E), and yeast-CEA (FIG. 2F).

FIG. 3A shows the discrete, non-overlapping CEA-526 and CEA-572 epitopeson the A3 loop of domain III of CEA.

FIGS. 3B-3C show that vaccination with rV/F-CEA/TRICOM or yeast-CEAinduces distinct cytokine profiles in response to in vitro stimulationwith two discrete CEA-specific epitopes, CEA-526 (FIG. 3B) and CEA-572(FIG. 3C).

FIGS. 4A-4D show that T-cell lines specific for the CEA-572 epitope frommice vaccinated with rV/F-CEA/TRICOM or yeast-CEA have distinct TCR Vαprofiles (Vα TCR repertoires of rV/F-CEA/TRICOM T-cell lines (blackbars) maintained in the presence of CEA-526 peptide (FIG. 4A) andCEA-572 peptide (FIG. 4B); Vα TCR repertoires of yeast-CEA T cell lines(white bars) maintained in the presence of CEA-526 peptide (FIG. 4C) andCEA-572 peptide (FIG. 4D)).

FIGS. 5A-5F show that epitope-specific T-cell lines generated from micevaccinated with rV/F-CEA/TRICOM or yeast-CEA have similar levels oflytic activity but unique avidity (FIGS. 5A and 5C show T cell linesgenerated from rV/F-CEA/TRICOM vaccination and specific for CEA-526peptide (FIG. 5A) and CEA-572 peptide (FIG. 5C); FIGS. 5B and 5D showT-cell lines generated from yeast-CEA vaccination and specific forCEA-526 peptide (FIG. 5B) and CEA-572 peptide (FIG. 5D); FIGS. 5E and 5Fshow T-cell lines specific for CEA-572 epitope, generated from micevaccinated with rV/F-CEA/TRICOM (FIG. 5E) or yeast-CEA (FIG. 5F)).

FIG. 6 shows that concurrently administering rV/F-CEA/TRICOM andyeast-CEA vaccines in an orthotopic pulmonary metastasis model increasesantitumor efficacy.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the concurrent use of twoimmunotherapeutic compositions (also referred to herein as vaccines) forthe induction of therapeutic immune responses and/or for the prevention,amelioration and/or treatment of disease, including, but not limited to,cancer and infectious disease. More specifically, using recombinantvirus immunotherapeutic vaccines and yeast-based immunotherapeuticvaccines, each targeting the same antigen or immunogenic domain thereof,both the vaccine vectors and the antigen are demonstrated herein to havea role in the induction of T-cell populations having both shared andunique cytokine responses, gene expression profiles, and T-cell receptorphenotypes. Accordingly, both the antigen and the vector play a role inthe induction of distinct T-cell populations. The studies presentedherein indicate that phenotypically and functionally distinct T-cellpopulations are induced by two vector platforms targeting the sameantigen. Finally, the inventors have demonstrated that the two vaccinescan be combined concurrently to improve therapeutic efficacy, showingthat the concurrent administration of the vaccines improved antitumorefficacy in the experiments described herein. These results indicate atherapeutic benefit from the concurrent administration of two distinctvector platforms targeting a single antigen, due to the induction of amore diverse T-cell population and improved therapeutic efficacy.

This study is believed to be the first to demonstrate effectiveconcurrent administration of two different vaccine vectors targeting thesame antigen. While it had been previously shown that each of thevaccines used in the studies described herein individually inducesimilar levels of CD4+ T-cell proliferation and CD8+ T-cell cytolyticactivity (Wansley et al., Clin Cancer Res, 2008 Jul. 1; 14(13):4316-25),the discovery that these vaccines induce phenotypically and functionallydistinct T-cell populations, and that the vaccines can be combinedconcurrently to substantially improve antitumor efficacy, was not knownor anticipated.

In contrast to prior studies showing that the functionality of T-cellpopulations induced by two different vaccine platforms (rV andrecombinant adenovirus) targeting the same antigen (OVA) did not differ(Millar et al., 2007, Cell Immunol 250(1-2):55-67), the presentinvention provides evidence that both the vector and the antigen affectthe functionality of the T-cell population induced by two differentimmunotherapeutic vaccines, one a virus-based immunotherapy composition(referred to herein as rV-CEA/TRICOM) and one a yeast-basedimmunotherapy composition (referred to herein as yeast-CEA). Comparingthe T-cell populations induced by both vaccines in terms of cytokineproduction, gene expression, and TCR profiling, the studies presentedherein found that rV-CEA/TRICOM induces a Th1 response and CD8+ T-cellswith a Tc1 phenotype, while yeast-CEA induces a mixed Th1/Th2 responseand CD8+ T-cells with a mixed Tc1/Tc2 phenotype (FIGS. 1 and 3).Up-regulation of genes involved in immune cell migration and TCRsignaling and T-cell proliferation was observed by both vaccines (Table1). Interestingly, although these vaccines modulate the expression ofgenes involved in various cellular pathways in an apparentdisadvantageous manner, antigen-specific immune responses or antitumorefficacy is not abrogated (Wansley et al., Clin Cancer Res 2008 Jul. 1;14(13):4316-25). It is also demonstrated herein that the T-cellpopulations induced by either vaccine have both shared and unique Vα andVβ TCR gene usage (FIG. 2) and that T-cell lines created from vaccinatedCEA-Tg mice, specific for one of two CEA epitopes demonstratedifferential avidity and antigen-specific cytolytic activity (FIG. 5).Taken together, these studies demonstrate that the two immunotherapeuticvaccines induce distinct T-cell populations, and the differences in thephenotype and function of these T-cell populations may be attributed toboth the vector and the antigen. These findings are applicable to theuse of any antigen in the context of the immunotherapy compositions.

The mode of antigen delivery by either vaccine platform may influencethe generation of these distinct responses. The mechanism by whichyeast-CEA predominantly activates the immune response is through theuptake of the CEA-expressing yeast and subsequent processing andpresentation of the CEA antigen by dendritic cells (DCs) (Bernstein etal., Vaccine 2008 Jan. 24; 26(4):509-21), while the rV/F-CEA/TRICOMvectors infect cells, inducing intracellular expression that allows theCEA antigen to be processed and presented (Hodge et al., Cancer Res 1999Nov. 15; 59(22):5800-7). Another difference observed is thatrV/F-CEA/TRICOM vaccination induces the production of CEA-specificantibody while yeast-CEA does not (data not shown). Such mechanisticdifferences in the activation of the cellular and humoral arms of theimmune system may also influence the vector- and antigen-specificinduction of distinct T-cell populations.

Surprisingly, the inventors discovered that administering two vaccinestargeting the same antigen induces distinct T-cell populations andresults in significantly higher antitumor immunity in a murineorthotopic pulmonary metastasis model (FIG. 6). This study also showedfor the first time that two vaccine platforms targeting the same antigencould be concurrently administered due to their induction of distinctT-cell populations. Furthermore, these data show that concurrentadministration of the two vaccines results in substantially increasedantitumor effects, due to the induction of a more diverse T-cellpopulation targeting the same antigen. These findings indicate thatconcurrently combining these vaccines can be utilized to increaseantigen-specific immunity. The concurrent use of immunotherapeuticvaccines as described herein induces a more diverse T-cell populationconsisting of T cells generated from both vaccines, making a diversifiedprime-boost schedule used prior to the invention unnecessary, althoughcombining a diversified prime-boost schedule with the concurrentadministration protocol described herein may further enhance aneffective immune response. The present invention maximizes the immuneresponse beginning with the initial vaccination by inducing a morediverse T-cell population that is then boosted and expanded in magnitudewith each subsequent vaccination. Such a strategy would be efficaciousin cancer patients as well as patients suffering from chronic infectiousdisease, because a more diverse T-cell population would be induced earlyin their treatment.

Accordingly, the present invention relates to the concurrent use of twoor more different immunotherapy compositions to induce therapeuticimmune responses against one or more antigens, and/or to prevent,ameliorate and/or treat a disease or condition, including cancer or aninfectious disease. In one embodiment, the two or more differentimmunotherapy compositions target the same antigen(s). In anotherembodiment, one of the two or more different immunotherapy compositionstargets one or more antigens, and the other of the immunotherapycompositions is provided as an adjuvant, without necessarily targetingan antigen, or without necessarily targeting the same antigen as theother composition (i.e., in one embodiment, the second compositiontargets a different antigen). In one embodiment, which may include anycombinations of the embodiments above, the two or more immunotherapycompositions are administered concurrently, but to different physicalsites in the patient. In another embodiment, which may include anycombination of the embodiments above, the two or more differentimmunotherapy compositions are administered concurrently and to thesame, or substantially adjacent, site in the patient.

The two or more different immunotherapy compositions used in the presentinvention are each capable, individually, of inducing an immuneresponse, and preferably, at least a cellular immune response, and morepreferably, a T cell-mediated cellular immune response. In one aspect,at least one of the compositions is capable of inducing a CD8+ and/or aCD4+ T cell-mediated immune response and more preferably, a CD8+ and aCD4+ T cell-mediated immune response. Preferably, all of thecompositions used concurrently according to the invention are capable ofinducing a CD8+ and/or a CD4+ T cell-mediated immune response, and morepreferably, a CD8+ and a CD4+ immune response. Optionally, at least oneof the compositions is capable of eliciting a humoral immune response.Preferably, the T cell-mediated immune response that is elicited by oneof the immunotherapy compositions is phenotypically and/or functionallydistinct in one or more aspects from the T cell-mediated immune responsethat is elicited by the other immunotherapy composition. In one aspect,the immunotherapeutic composition has one or more of the followingcharacteristics: (a) stimulates one or more pattern recognitionreceptors effective to activate an antigen presenting cell; (b)upregulates adhesion molecules, co-stimulatory molecules, and MHC classI and/or class II molecules on antigen presenting cells; (c) inducesproduction of proinflammatory cytokines by antigen presenting cells; (d)induces production of Th1-type cytokines by T cells; (e) inducesproduction of Th17-type cytokines by T cells; (f) inhibits ordownregulates Treg; and/or (g) elicits MHC Class I and/or MHC Class II,antigen-specific immune responses. Suitable immunotherapeuticcompositions can include yeast-based immunotherapy compositions,viral-based immunotherapy compositions, antibody-based immunotherapycompositions, DNA immunotherapy compositions, subunit vaccines, and anycomponents or adjuvants useful for stimulating or modulating an immuneresponse, such as TLR agonists, cytokines, immune potentiators, andother agents, and any combinations thereof, many of which are describedin more detail below. In one aspect, immunotherapy compositions to beused in the present invention include recombinant virus-basedcompositions and yeast-based compositions (described in detail below).

Virus-Based Immunotherapy Compositions

One aspect of the invention relates to a recombinant virus-basedimmunotherapy composition (which phrase may be used interchangeably with“virus-based immunotherapy product”, “virus-based composition”,“virus-based immunotherapeutic”, “virus-based vaccine”, “immunotherapycomposition comprising or including a recombinant virus or recombinantviral vector”, or any similar derivation of these phrases). As usedherein, the phrase virus-based immunotherapy composition” refers to acomposition that includes a viral vector component (e.g., a recombinantvirus or portion thereof effective to constitute a viral vector) andthat elicits an immune response sufficient to achieve at least onetherapeutic benefit in a subject. Virus-based immunotherapy compositionsand methods of making and generally using the same, are described indetail, for example, in U.S. Pat. Nos. 6,045,802, 6,893,869, 6,548,068,and 6,969,609, each of which is incorporated herein by reference in itsentirety. In one aspect, a virus-based immunotherapy composition usefulin the invention is capable of inducing a CD8+ and/or a CD4+ Tcell-mediated immune response and in one aspect, a CD8+ and a CD4+ Tcell-mediated immune response, and in one aspect, a humoral immuneresponse. A virus-based immunotherapy composition useful in the presentinvention can, for example, elicit an immune response in an individualsuch that the individual is treated for the disease or condition, orsuch that symptoms resulting from the disease or condition arealleviated or treated.

A virus-based immunotherapy composition typically comprises a viralvector comprising a virus genome or portions thereof (e.g., arecombinant virus) and a nucleic acid sequence encoding at least oneantigen(s) from a disease causing agent or disease state (e.g., a cancerantigen(s), infectious disease antigen(s), and/or at least oneimmunogenic domain thereof). In some embodiments, a virus-basedimmunotherapy composition further includes at least one viral vectorcomprising one or more nucleic acid sequences encoding one or moreimmunostimulatory molecule(s). In some embodiments, the genes encodingimmunostimulatory molecules and antigens are inserted into the sameviral vector (the same recombinant virus).

Viruses that may be used in such immunotherapy compositions of thepresent invention are any viruses that infect cells, inducingintracellular expression of the antigen carried by the virus that allowsthe antigen to be processed and presented. Preferred among these virusesare those that induce a Th1 response and CD8+ T-cells with a Tc1phenotype. Viruses that may be used in a composition of the inventioninclude viruses in which a portion of the genome can be deleted tointroduce new genes without destroying infectivity of the virus.

Parental viruses (i.e., viruses from which viral vectors/recombinantviruses are produced, derived, based, etc.) useful in the production ofviral vectors of the invention include, but are not limited to,poxvirus, Herpes virus, adenovirus, alphavirus, retrovirus,picornavirus, baculovirus, and iridovirus. Poxviruses (members of thefamily Poxviridae) having utility in the present invention includereplicating and non-replicating vectors. Such poxviruses include, butare not limited to, orthopox (genus Orthopoxvirus) such as vacciniavirus (Perkus et al., Science 229:981-984, 1985; Kaufman et al., Int. J.Cancer 48:900-907, 1991, Moss, Science 252:1662, 1991), raccoon pox,rabbit pox and the like, avipox (genus Avipoxvirus) including fowlpoxvirus, suipox (genus Suipoxvirus), capripox (genus Capripoxvirus) andthe like. Poxviruses may be selected from the group ofvaccinia-Copenhagen, vaccinia-Wyeth strain, highly attenuated vacciniavirus (vaccinia-MVA strain), modified vaccinia Ankara (Sutter and Moss,Proc. Nat'l Acad. Sci. U.S.A. 89:10847-10851; Sutter et al., Virology1994), NYVAC, TROVAC, canarypox, ALVAC (Baxby and Paoletti, Vaccine10:8-9, 1992; Rinns, M. M. et al., (Eds) Recombinant Poxviruses CRCPress, Inc., Boca Raton 1992; Paoletti, E., Proc. Nat'l Acad. Sci. USA93:11349-11353, 1996), swinepox, and the like. A derivative of thevaccinia-Wyeth strain includes but is not limited to vTBC33 which lacksa functional K1L gene. In yet another embodiment, the virus is Dry-Vaxavailable as a smallpox vaccine from the Centers for Disease Control,Atlanta, Ga. In one embodiment, the recombinant vector is a vacciniavirus. In another embodiment, the recombinant vector is from fowlpoxvirus. One strain of fowlpox virus, for example, is PDXVAC-TC(Schering-Plough Corporation).

Recombinant poxviruses having utility in the present invention have anumber of attributes, including (i) efficient delivery of genes tomultiple cell types, including antigen presenting cells (APC) and tumorcells; (ii) high levels of protein expression; (iii) optimalpresentation of antigens to the immune system; (iv) the ability toelicit cell-mediated immune responses as well as antibody responses; (v)transient, rather than permanent, genetic modification of cells, and(vi) the ability to use combinations of poxviruses from differentgenera, as they are not immunologically cross-reactive.

Recombinant vaccinia (rV, rMVA) and recombinant fowlpox (rF) viruses aretwo exemplary viruses for use in the virus-based immunotherapycompositions of the invention. Recombinant vaccinia and fowlpox virusescontaining murine B7-1, ICAM-1, and LFA-3 genes as well as the human CEAgene (rV/F-CEA/TRICOM) have been described (Hodge et al., Cancer Res1999 Nov. 15; 59(22):5800-7; Grosenbach et al., Cancer Res 2001 Jun. 1;61(11):4497-505; and Hodge et al., Cancer Res 2003 Nov. 15;63:7942-7949), each of which is incorporated herein by reference in itsentirety. The murine GM-CSF-expressing rF virus (rF-GM-CSF) has beenpreviously described (Kass et al., Cancer Res 2001 Jan. 1;61(1):206-14). For purposes of the present invention, a vaccinedesignated “rV/F-CEA/TRICOM” refers to a complete vaccine protocol thatincludes a priming with rV-CEA/TRICOM (recombinant vaccinia containingpolynucleotides encoding B7-1, ICAM-1, LFA-3 and CEA) admixed withrF-GM-CSF (recombinant fowl pox containing a gene encoding GM-CSF) whichis boosted every 7 days with rF-CEA/TRICOM recombinant fowlpoxcontaining polynucleotides encoding B7-1, ICAM-1, LFA-3 and CEA) admixedwith rF-GM-CSF. It is to be understood that CEA is just one example of asuitable antigen (in this case, a tumor-associated or cancer antigen) tobe used in connection with one or more immunotherapy compositions of theinvention.

The viral vector of the present invention comprises at least oneexpression control element operably linked to the nucleic acid sequence.The expression control elements are inserted in the vector to controland regulate the expression of the nucleic acid sequence (Ausubel et al,1987, in “Current Protocols in Molecular Biology, John Wiley and Sons,New York, N.Y.). Expression control elements are known in the art andinclude promoters. Promoters useful in the present invention arepoxviral promoters as are known in the art which include, but are notlimited to, 30K, 13, sE/L, 7.5K, 40K, and Cl. The nucleic acid sequenceof the 30K promoter is disclosed in GenBank Accession No. M35027 at basenumbers 28,012 through 28,423 (antisense). The nucleic acid sequence of13 is disclosed in GenBank Accession No. J03399 at base numbers 1100through 1301 (antisense). The nucleic acid sequence of the 7.5K promoteris disclosed in GenBank Accession No. M35027 at base numbers 186550through 186680. The nucleic acid sequence of the 40K promoter isdisclosed in GenBank Accession No. M13209 at base numbers 9700 through9858 (antisense). The nucleic acid sequence of the Cl promoter isdisclosed in GenBank Accession No. M59027 at base numbers 1 through 242and in U.S. Pat. No. 5,093,258. The sequence of the sE/L promoter isknown in the art. Other poxvirus promoters may be used, such as, thosedescribed by Davison and Moss (J. Mol. Biol. 210:749-769. (1989). Any ofthese promoters can be synthesized by using standard methods in the art.The selection of an appropriate promoter is based on its timing andlevel of expression. Early or early/late promoters are used in oneaspect. In one embodiment, the promoter or combination of promotersutilized allow for optimal expression of each antigen and/orcostimulatory molecule in an infected host to provide a synergisticimmune response. In one embodiment, each nucleic acid molecule encodingan antigen or costimulatory molecule is controlled by a separate anddistinct promoter.

As used herein with any immunotherapy composition described for use inthe present invention, immunostimulatory molecules include, but are notlimited to, cytokines and costimulatory molecules. For example,cytokines can include, but are not limited to, tumor necrosis factor-α(TNF-α), interleukin-6 (IL-6), granulocyte-macrophage-colony stimulatingfactor (GM-CSF), interferon-γ (IFN-γ), IFN-α, IFN-λ, interleukin-12(IL-12), RANTES, and interleukin-2 (IL-2). Costimulatory moleculesinclude, but are not limited to, B7.1 (also referred to herein as B7-1),B7.2 (also referred to herein as B7-2), ICAM-1, LFA-3, 4-1BBL, CD59,CD40, CD40L and CD70. Immunostimulatory molecules can be provided in animmunotherapy composition of the invention alone and/or in a variety ofcombinations, and can be expressed by one or more viral vectors or othervaccine vectors. In one aspect, the recombinant vector of the presentinvention comprises genes encoding at least three costimulatorymolecules for synergistic enhancement of immune responses which is notobtainable by the use of a single or a double costimulatory molecule.Genes encoding various combinations of costimulatory molecules are anelement of the invention for use in the recombinant vector and mayinclude such combinations as: B7.1, B7.2, ICAM-1, and LFA-3; B7.1,ICAM-1, and LFA-3; B7.1, B7.2, ICAM-1, and 4-1BBL; B7.1, B7.2, ICAM-1,LFA-3, and 4-1BBL; CD59 and VCAM-1; and B7.1 and B7.2; CD59, CD40,4-BBL, CD70 and VCAM-1, B7.1, B7.2; OX-40L, 4-1BBL; depending on thedesired immune response and the disease or condition to be treated.

B7.1 (CD80) is a natural ligand for the T-cell antigen, CD28, mediatingT- and B-cell adhesion. B7.1 is expressed on activated B-cells andgamma-interferon-stimulated monocytes. The binding of CD80 to CD28 andCTLA-4 provides a co-stimulatory signal to T-cells and leads toupregulated lymphokine production. B7.2 (CD86) is an alternative ligandfor CD28 and CTLA-4.

ICAM-1 (Inter-Cellular Adhesion Molecule 1), also known as CD54, is aleukocyte- and endothelial-associated intercellular adhesion moleculeand is a ligand for LFA-1 (integrin), a leukocyte receptor. ICAM-1 isupregulated upon cytokine stimulation and is involved in cellularinteractions, leukocyte endothelial transmigration, and signalingrelated to recruitment of proinflammatory immune cells.

LFA-3 (lymphocyte function associated antigen-3), or CD58, is anadhesion molecule expressed by antigen presenting cells (APCs),mediating a costimulatory signal through CD2, an adhesion molecule foundon T cells and natural killer (NK) cells. This interaction promotesintercellular adhesion and the activation of T cells.

4-1BBL is expressed on activated antigen presenting cells (APCs) and isthe ligand for 4-1BB, a costimulatory member of the tumor necrosisfactor receptor family expressed on activated CD4 and CD8 T cells. Theinteraction of these molecules can enhance T cell proliferation andsurvival, and expand and activate CD8+ T cell memory (Bukczynski et al.,Proc. Natl. Acad. Sci. U.S.A. 2004, 101(5):1291-6).

CD59 is a complement regulatory protein. Viruses, including vacciniavirus, vaccinia incorporate host cell CD59 into their own viral envelopeto prevent lysis by complement (Bohana-Kashtan et al., 2004, Mol.Immunol. 41 (6-7): 583-97).

VCAM-1 (vascular cell adhesion molecule-1), or CD106, is an adhesionmolecule expressed by endothelial cells and mediates the adhesion oflymphocytes, monocytes, eosinophils, and basophils to vascularendothelium. It also functions in leukocyte-endothelial cell signaltransduction.

CD40 is a costimulatory protein expressed by antigen presenting cells(APCs). Binding of CD40 to its natural ligand, CD40L, on T cells,activates the APC and initiates a signaling cascade that mediates avariety of immune and inflammatory responses.

CD70 is a ligand for CD27, which is a receptor required for thegeneration and maintenance of long-term T cell immunity.

OX40-L is expressed on expressed on the surface of activated B cells, Tcells, dendritic cells and endothelial cells and binds to its naturalreceptor, OX40, which is primarily expressed by activated CD4+ T cells,and therefore, this interaction is related to T cell activation.

GM-CSF (granulocyte macrophage colony stimulating factor) is a cytokinethat functions as a white blood cell growth factor produced by T cells,macrophages, and other cells. GM-CSF stimulates stem cells to producegranulocytes (neutrophils, eosinophils, and basophils) and monocytes,and is thus part of the immune system activation and developmentprocess.

Simultaneous production of an immunostimulatory molecule and theantigen(s) at the site of virus replication/infection (in any case, thesite of antigen production) enhances the generation of specificeffectors. Dependent upon the specific immunostimulatory molecules,different mechanisms might be responsible for the enhancedimmunogenicity: augmentation of help signal (IL-2), recruitment ofprofessional APC (GM-CSF), increase in CTL frequency (IL-2), and/oreffect on antigen processing pathway and MHC expression (IFN-γ andTNFα). In some cases, it may be beneficial to produce a recombinantvirus comprising more than one antigen of interest for the purpose ofhaving a multivalent vaccine.

In one embodiment, a pharmaceutical composition comprises a recombinantvirus (e.g., a poxvirus) containing nucleic acid molecules encodingmultiple costimulatory molecules in a pharmaceutically acceptablecarrier. The recombinant virus may further comprise a nucleic acidsequence encoding at least one antigen or immunogenic domain thereof oralternatively, a second recombinant virus (e.g., a poxvirus) may beprovided encoding at least one antigen or immunogenic domain thereof.

In one embodiment, a virus-based immunotherapy composition useful in thepresent invention includes a pharmaceutical composition comprising arecombinant virus (e.g., a recombinant poxvirus) comprising a nucleicacid sequence encoding B7.1 or B7.2, a nucleic acid sequence encodingICAM-1, and a nucleic acid sequence encoding LFA-3 and apharmaceutically acceptable carrier. In addition to the B7, ICAM-1,LFA-3 construct, the recombinant virus of the pharmaceutical compositionmay additionally comprise a nucleic acid sequence encoding at least oneantigen or immunogenic domain thereof, or alternatively, the nucleicacid sequence encoding at least one antigen or immunogenic domainthereof may be provided in the composition by a second recombinantvirus.

In one embodiment, the composition may also comprise exogenously addedimmunostimulatory molecules as are known in the art including, but notlimited to, the costimulatory molecules B7, ICAM-1, LFA-3, 4-1BBL, CD59,CD40, CD70, VCAM-1, OX-40L and/or antibodies that bind to suchimmunostimulatory molecules, and/or agonists or antagonists of suchimmunostimulatory molecules, and/or cytokines and chemokines includingbut not limited to IL-2, GM-CSF, TNF-α, IFN-γ, IFN-α, IFN-λ, IL-12,RANTES, MIP-la, Flt-3L (U.S. Pat. Nos. 5,554,512; 5,843,423) and thelike, for additional synergy or enhancement of an immune response. Thecytokines and chemokines themselves may be provided in the compositionor, alternatively, the cytokines and chemokines may be provided by arecombinant viral vector encoding the cytokine or chemokine.

In one embodiment of the present invention, a recombinant poxvirus isprovided comprising a nucleic acid sequence encoding LFA-3 or functionalportion thereof under control of a 30K poxviral promoter, a nucleic acidsequence encoding ICAM-1 or portion thereof under control of an 13poxviral promoter, and a nucleic acid sequence encoding B7.1 or portionthereof under control of an sE/L poxviral promoter. The recombinantpoxvirus may further provide a nucleic acid sequence encoding at leastone antigen or immunogenic domain thereof.

In another embodiment of the present invention, a recombinant poxvirusis provided comprising a nucleic acid sequence encoding B7.1 undercontrol of a sE/L poxviral promoter, a nucleic acid sequence encodingLFA-3 or portion thereof under control of the 13 poxviral promoter, anda nucleic acid sequence encoding ICAM-1 or portion thereof under controlof the 7.5K poxvirus promoter. Optionally the construct furthercomprises a nucleic acid sequence encoding at least antigen orimmunogenic domain thereof.

In an embodiment of the invention, a recombinant fowlpox virus comprisesa nucleic acid sequence encoding B7.1 or portion thereof under controlof the sE/L poxviral promoter, a nucleic acid sequence encoding LFA-3 orportion thereof under control of the 13 poxviral promoter, and a nucleicacid sequence encoding ICAM-1 or portion thereof under control of the7.5K poxviral promoter. A recombinant fowlpox virus may further comprisea nucleic acid sequence encoding a target antigen under control of apoxviral promoter such as the 40K poxviral promoter.

In some embodiments of the invention, the recombinant virus-basedimmunotherapy compositions do not express an antigen, although in such acase, the recombinant viral vector(s) of such compositions preferablystill express one or more immunostimulatory molecules as describedelsewhere herein. In this embodiment of the invention, the antigen isprovided by one or more different immunotherapy compositions asdescribed herein, such as the yeast-based immunotherapy composition ofthe invention. Because each of the immunotherapy compositions used inthe invention may provide some unique “danger signals” or costimulatorysignals to the immune system, it may be sufficient to provide thecontributions of the vectors while only one of the compositionscomprises an antigen.

The present invention further provides methods of generating recombinantviruses comprising nucleic acid sequences encoding antigens and/ormultiple costimulatory molecules. One method of generation ofrecombinant poxviruses is accomplished via homologous recombination invivo between parental poxvirus genomic DNA and a plasmid vector thatcarries the heterologous sequences to be inserted, as disclosed in U.S.Pat. No. 5,093,258. Plasmid vectors for the insertion of foreignsequences into poxviruses are constructed by standard methods ofrecombinant DNA technology. The plasmid vectors contain one or morechimeric foreign genes, each comprising a poxvirus promoter linked to aprotein coding sequence, flanked by viral sequences from a non-essentialregion of the poxvirus genome. The plasmid is transfected into cellsinfected with the parental poxvirus using art accepted transfectionmethods, and recombination between poxvirus sequences on the plasmid andthe corresponding DNA in the parental viral genome results in theinsertion into the viral genome of the chimeric foreign genes from theplasmid. Recombinant viruses are selected and purified using any of avariety of selection or screening systems as are known in the art.Insertion of the foreign genes into the vaccinia genome is confirmed bypolymerase chain reaction (PCR) analysis. Expression of the foreigngenes is demonstrated by Western blot analysis. An alternative method ofgeneration of recombinant poxviruses is accomplished by direct ligation(Pleiderer et al., J. Gen. Virol. 76:2957-2962, 1995; Merchlinsky etal., Virol. 238:444-451, 1997).

Yeast-Based Immunotherapy Compositions

In one embodiment of the invention, the invention includes the use of atleast one “yeast-based immunotherapeutic composition” (which phrase maybe used interchangeably with “yeast-based immunotherapy product”,“yeast-based composition”, “yeast-based immunotherapeutic”, “yeast-basedvaccine”, “immunotherapy composition comprising a yeast vehicle”, or anysimilar derivation of these phrases). As used herein, the phrase“yeast-based immunotherapy composition” refers to a composition thatincludes a yeast vehicle component and that elicits an immune responsesufficient to achieve at least one therapeutic benefit in a subject.More particularly, a yeast-based immunotherapeutic composition is acomposition that includes a yeast vehicle component and can elicit orinduce an immune response, such as a cellular immune response, includingwithout limitation a T cell-mediated cellular immune response. In oneaspect, a yeast-based immunotherapy composition useful in the inventionis capable of inducing a CD8+ and/or a CD4+ T cell-mediated immuneresponse and in one aspect, a CD8+ and a CD4+ T cell-mediated immuneresponse. Optionally, a yeast-based immunotherapy composition is capableof eliciting a humoral immune response. A yeast-based immunotherapycomposition useful in the present invention can, for example, elicit animmune response in an individual such that the individual is treated forthe disease or condition, or such that symptoms resulting from thedisease or condition are alleviated or treated.

Typically, a yeast-based immunotherapy composition includes a yeastvehicle and at least one antigen or immunogenic domain thereof expressedby, attached to, or mixed with the yeast vehicle. In some embodiments,the antigen or immunogenic domain thereof is provided as a fusionprotein. In one aspect of the invention, fusion protein can include twoor more antigens. In one aspect, the fusion protein can include two ormore immunogenic domains of one or more antigens, or two or moreepitopes of one or more antigens. A TARMOGEN® is one non-limitingexample of a yeast-based immunotherapy composition that is useful in thepresent invention. A TARMOGEN® (TARgeted MOlecular immunoGEN,Globelmmune, Inc., Louisville, Colo.) generally refers to a yeastvehicle expressing one or more heterologous antigens extracellularly (onits surface), intracellularly (internally or cytosolically) or bothextracellularly and intracellularly.

Yeast-based immunotherapy compositions, and methods of making andgenerally using the same, are described in detail, for example, in U.S.Pat. Nos. 5,830,463, 7,083,787, 7,465,454, U.S. Patent Publication2007-0224208, U.S. Patent Publication No. US 2008-0003239, and in Stubbset al., Nat. Med. 7:625-629 (2001), Lu et al., Cancer Research64:5084-5088 (2004), and in Bernstein et al., Vaccine 2008 Jan. 24;26(4):509-21, each of which is incorporated herein by reference in itsentirety. These yeast-based immunotherapeutic products have been shownto elicit immune responses, including cellular and humoral immuneresponses. Yeast-based immunotherapeutic products are capable of killingtarget cells expressing a variety of antigens in vivo, in a variety ofanimal species, and do so via antigen-specific, CD8+ and/or CD4+mediated immune responses. Additional studies have shown that yeast areavidly phagocytosed by and directly activate dendritic cells which thenpresent yeast-associated proteins to CD4+ and CD8+ T cells in a highlyefficient manner. See, e.g., Stubbs et al. Nature Med. 5:625-629 (2001)and U.S. Pat. No. 7,083,787.

In any of the yeast-based immunotherapy compositions used in the presentinvention, the following aspects related to the yeast vehicle areincluded in the invention. According to the present invention, a yeastvehicle is any yeast cell (e.g., a whole or intact cell) or a derivativethereof (see below) that can be used in conjunction with one or moreantigens, immunogenic domains thereof or epitopes thereof in atherapeutic composition of the invention, or in one aspect, the yeastvehicle can be used alone or as an adjuvant. The yeast vehicle cantherefore include, but is not limited to, a live intact yeastmicroorganism (i.e., a yeast cell having all its components including acell wall), a killed (dead) or inactivated intact yeast microorganism,or derivatives thereof including: a yeast spheroplast (i.e., a yeastcell lacking a cell wall), a yeast cytoplast (i.e., a yeast cell lackinga cell wall and nucleus), a yeast ghost (i.e., a yeast cell lacking acell wall, nucleus and cytoplasm), a subcellular yeast membrane extractor fraction thereof (also referred to as a yeast membrane particle andpreviously as a subcellular yeast particle), any other yeast particle,or a yeast cell wall preparation.

Yeast spheroplasts are typically produced by enzymatic digestion of theyeast cell wall. Such a method is described, for example, in Franzusoffet al., 1991, Meth. Enzymol. 194, 662-674., incorporated herein byreference in its entirety.

Yeast cytoplasts are typically produced by enucleation of yeast cells.Such a method is described, for example, in Coon, 1978, Natl. CancerInst. Monogr. 48, 45-55 incorporated herein by reference in itsentirety.

Yeast ghosts are typically produced by resealing a permeabilized orlysed cell and can, but need not, contain at least some of theorganelles of that cell. Such a method is described, for example, inFranzusoff et al., 1983, 1 Biol. Chem. 258, 3608-3614 and Bussey et al.,1979, Biochim. Biophys. Acta 553, 185-196, each of which is incorporatedherein by reference in its entirety.

A yeast membrane particle (subcellular yeast membrane extract orfraction thereof) refers to a yeast membrane that lacks a naturalnucleus or cytoplasm. The particle can be of any size, including sizesranging from the size of a natural yeast membrane to microparticlesproduced by sonication or other membrane disruption methods known tothose skilled in the art, followed by resealing. A method for producingsubcellular yeast membrane extracts is described, for example, inFranzusoff et al., 1991, Meth. Enzymol. 194, 662-674. One may also usefractions of yeast membrane particles that contain yeast membraneportions and, when the antigen or other protein was expressedrecombinantly by the yeast prior to preparation of the yeast membraneparticles, the antigen or other protein of interest. Antigens or otherproteins of interest can be carried inside the membrane, on eithersurface of the membrane, or combinations thereof (i.e., the protein canbe both inside and outside the membrane and/or spanning the membrane ofthe yeast membrane particle). In one embodiment, a yeast membraneparticle is a recombinant yeast membrane particle that can be an intact,disrupted, or disrupted and resealed yeast membrane that includes atleast one desired antigen or other protein of interest on the surface ofthe membrane or at least partially embedded within the membrane.

An example of a yeast cell wall preparation is isolated yeast cell wallscarrying an antigen on its surface or at least partially embedded withinthe cell wall such that the yeast cell wall preparation, whenadministered to an animal, stimulates a desired immune response againsta disease target.

Any yeast strain can be used to produce a yeast vehicle of the presentinvention. Yeast are unicellular microorganisms that belong to one ofthree classes: Ascomycetes, Basidiomycetes and Fungi Imperfecti. Oneconsideration for the selection of a type of yeast for use as an immunemodulator is the pathogenicity of the yeast. In one embodiment, theyeast is a non-pathogenic strain such as Saccharomyces cerevisiae. Theselection of a non-pathogenic yeast strain minimizes any adverse effectsto the individual to whom the yeast vehicle is administered. However,pathogenic yeast may be used if the pathogenicity of the yeast can benegated by any means known to one of skill in the art (e.g., mutantstrains). In accordance with one aspect of the present invention,nonpathogenic yeast strains are used.

Genera of yeast strains that may be used in the invention include butare not limited to Saccharomyces, Candida (which can be pathogenic),Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula,Schizosaccharomyces and Yarrowia. In one aspect, yeast genera areselected from Saccharomyces, Candida, Hansenula, Pichia orSchizosaccharomyces, and in one aspect, Saccharomyces is used. Speciesof yeast strains that may be used in the invention include but are notlimited to Saccharomyces cerevisiae, Saccharomyces carlsbergensis,Candida albicans, Candida kefyr, Candida tropicalis, Cryptococcuslaurentii, Cryptococcus neoformans, Hansenula anomala, Hansenulapolymorpha, Kluyveromyces Kluyveromyces lactis, Kluyveromyces marxianusvar. lactis, Pichia pastoris, Rhodotorula rubra, Schizosaccharomycespombe, and Yarrowia lipolytica. It is to be appreciated that a number ofthese species include a variety of subspecies, types, subtypes, etc.that are intended to be included within the aforementioned species. Inone aspect, yeast species used in the invention include S. cerevisiae,C. albicans, H. polymorpha, P. pastoris and S. pombe. S. cerevisiae isuseful due to it being relatively easy to manipulate and being“Generally Recognized As Safe” or “GRAS” for use as food additives(GRAS, FDA proposed Rule 62FR18938, Apr. 17, 1997). One embodiment ofthe present invention is a yeast strain that is capable of replicatingplasmids to a particularly high copy number, such as a S. cerevisiaecir° strain. The S. cerevisiae strain is one such strain that is capableof supporting expression vectors that allow one or more targetantigen(s) and/or antigen fusion protein(s) and/or other proteins to beexpressed at high levels. In addition, any mutant yeast strains can beused in the present invention, including those that exhibit reducedpost-translational modifications of expressed target antigens or otherproteins, such as mutations in the enzymes that extend N-linkedglycosylation.

In one embodiment, a yeast vehicle of the present invention is capableof fusing with the cell type to which the yeast vehicle andantigen/agent is being delivered, such as a dendritic cell ormacrophage, thereby effecting particularly efficient delivery of theyeast vehicle, and in many embodiments, the antigen(s) or other agent,to the cell type. As used herein, fusion of a yeast vehicle with atargeted cell type refers to the ability of the yeast cell membrane, orparticle thereof, to fuse with the membrane of the targeted cell type(e.g., dendritic cell or macrophage), leading to syncytia formation. Asused herein, a syncytium is a multinucleate mass of protoplasm producedby the merging of cells. A number of viral surface proteins (includingthose of immunodeficiency viruses such as HIV, influenza virus,poliovirus and adenovirus) and other fusogens (such as those involved infusions between eggs and sperm) have been shown to be able to effectfusion between two membranes (i.e., between viral and mammalian cellmembranes or between mammalian cell membranes). For example, a yeastvehicle that produces an HIV gp120/gp41 heterologous antigen on itssurface is capable of fusing with a CD4+ T-lymphocyte. It is noted,however, that incorporation of a targeting moiety into the yeastvehicle, while it may be desirable under some circumstances, is notnecessary. In the case of yeast vehicles that express antigensextracellularly, this can be a further advantage of the yeast vehiclesof the present invention. In general, yeast vehicles useful in thepresent invention are readily taken up by dendritic cells (as well asother cells, such as macrophages).

In some embodiments of the invention, the yeast-based immunotherapycomposition includes at least one antigen, immunogenic domain thereof,or epitope thereof. The antigens contemplated for use in this inventioninclude any antigen against which it is desired to elicit an immuneresponse (described in more detail below).

In some embodiments of the invention, the yeast-based immunotherapycompositions do not express or otherwise contain or display an antigen,although in this case, the yeast vehicle may optionally express one ormore immunostimulatory molecules. In this embodiment of the invention,the antigen is provided by one or more different immunotherapycompositions as described herein, such as a virus-based immunotherapycomposition. Because each of the immunotherapy compositions used in theinvention may provide some unique “danger signals” or costimulatorysignals to the immune system, it may be sufficient to provide thecontributions of the vectors while only one of the compositionscomprises an antigen.

According to the present invention, the term “yeast vehicle-antigencomplex” or “yeast-antigen complex” is used generally to describe anyassociation of a yeast vehicle with an antigen, and can be usedinterchangeably with “yeast-based immunotherapy composition” when suchcomposition is used to elicit an immune response as described above.Such association includes expression of the antigen by the yeast (arecombinant yeast), introduction of an antigen into a yeast, physicalattachment of the antigen to the yeast, and mixing of the yeast andantigen together, such as in a buffer or other solution or formulation.These types of complexes are described in detail below.

In one embodiment, a yeast cell (e.g., a whole yeast) used to preparethe yeast vehicle or that is the yeast vehicle is transfected with aheterologous nucleic acid molecule encoding a protein (e.g., the antigenor agent) such that the protein is expressed by the yeast cell. Such ayeast is also referred to herein as a recombinant yeast or a recombinantyeast vehicle. The yeast cell can then be administered, or the yeastcell can be killed, or it can be derivatized such as by formation ofyeast spheroplasts, cytoplasts, ghosts, or subcellular particles. Yeastspheroplasts can also be directly transfected with a recombinant nucleicacid molecule (e.g., the spheroplast is produced from a whole yeast, andthen transfected) in order to produce a recombinant spheroplast thatexpresses an antigen or other protein.

In one aspect, a yeast cell or yeast spheroplast used to prepare theyeast vehicle is transfected with a recombinant nucleic acid moleculeencoding the antigen(s) or other protein such that the antigen or otherprotein is recombinantly expressed by the yeast cell or yeastspheroplast. In this aspect, the yeast cell or yeast spheroplast thatrecombinantly expresses the antigen(s) or other protein is used toproduce a yeast vehicle comprising a yeast cytoplast, a yeast ghost, ora yeast membrane particle or yeast cell wall particle, or fractionthereof.

A number of antigens and/or other proteins to be produced by a yeastvehicle of the present invention is any number of antigens and/or otherproteins that can be reasonably produced by a yeast vehicle, andtypically ranges from at least one to at least about 6 or more,including from about 2 to about 6 heterologous antigens and or otherproteins.

Expression of an antigen or other protein in a yeast vehicle of thepresent invention can be accomplished using techniques known to thoseskilled in the art. Briefly, in one aspect, a nucleic acid moleculeencoding at least one desired antigen or other protein is inserted intoan expression vector in such a manner that the nucleic acid molecule isoperatively linked to a transcription control sequence in order to becapable of effecting either constitutive or regulated expression of thenucleic acid molecule when transformed into a host yeast cell. Nucleicacid molecules encoding one or more antigens and/or other proteins canbe on one or more expression vectors operatively linked to one or moreexpression control sequences. Particularly important expression controlsequences are those which control transcription initiation, such aspromoter and upstream activation sequences. Any suitable yeast promotercan be used in the present invention and a variety of such promoters areknown to those skilled in the art. Promoters for expression inSaccharomyces cerevisiae include, but are not limited to, promoters ofgenes encoding the following yeast proteins: alcohol dehydrogenase I(ADH1) or II (ADH2), CUP1, phosphoglycerate kinase (PGK), triosephosphate isomerase (TPI), translational elongation factor EF-1 alpha(TEF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also referredto as TDH3, for triose phosphate dehydrogenase), galactokinase (GAL1),galactose-1-phosphate uridyl-transferase (GALT), UDP-galactose epimerase(GAL10), cytochrome cl (CYC1), Sec? protein (SECT) and acid phosphatase(PHOS), including hybrid promoters such as ADH2/GAPDH and CYC1/GAL10promoters, and including the ADH2/GAPDH promoter, which is induced whenglucose concentrations in the cell are low (e.g., about 0.1 to about 0.2percent), as well as the CUP1 promoter and the TEF2 promoter. Likewise,a number of upstream activation sequences (UASs), also referred to asenhancers, are known. Upstream activation sequences for expression inSaccharomyces cerevisiae include, but are not limited to, the UASs ofgenes encoding the following proteins: PCK1, TPI, TDH3, CYC1, ADH1,ADH2, SUC2, GAL1, GALT and GAL10, as well as other UASs activated by theGAL4 gene product, with the ADH2 UAS being used in one aspect. Since theADH2 UAS is activated by the ADR1 gene product, it may be preferable tooverexpress the ADR1 gene when a heterologous gene is operatively linkedto the ADH2 UAS. Transcription termination sequences for expression inSaccharomyces cerevisiae include the termination sequences of theα-factor, GAPDH, and CYC1 genes.

Transcription control sequences to express genes in methyltrophic yeastinclude the transcription control regions of the genes encoding alcoholoxidase and formate dehydrogenase.

Transfection of a nucleic acid molecule into a yeast cell according tothe present invention can be accomplished by any method by which anucleic acid molecule administered into the cell and includes, but isnot limited to, diffusion, active transport, bath sonication,electroporation, microinjection, lipofection, adsorption, and protoplastfusion. Transfected nucleic acid molecules can be integrated into ayeast chromosome or maintained on extrachromosomal vectors usingtechniques known to those skilled in the art. Examples of yeast vehiclescarrying such nucleic acid molecules are disclosed in detail herein. Asdiscussed above, yeast cytoplast, yeast ghost, and yeast membraneparticles or cell wall preparations can also be produced recombinantlyby transfecting intact yeast microorganisms or yeast spheroplasts withdesired nucleic acid molecules, producing the antigen therein, and thenfurther manipulating the microorganisms or spheroplasts using techniquesknown to those skilled in the art to produce cytoplast, ghost orsubcellular yeast membrane extract or fractions thereof containingdesired antigens or other proteins.

In one aspect of the invention, the yeast vehicle is manipulated suchthat the antigen is expressed or provided by delivery or translocationof an expressed protein product, partially or wholly, on the surface ofthe yeast vehicle (extracellular expression). One method foraccomplishing this aspect of the invention is to use a spacer arm forpositioning one or more protein(s) on the surface of the yeast vehicle.For example, one can use a spacer arm to create a fusion protein of theantigen(s) or other protein of interest with a protein that targets theantigen(s) or other protein of interest to the yeast cell wall. Forexample, one such protein that can be used to target other proteins is ayeast protein (e.g., cell wall protein 2 (cwp2), Aga2, Pir4 or Flo1protein) that enables the antigen(s) or other protein to be targeted tothe yeast cell wall such that the antigen or other protein is located onthe surface of the yeast. Proteins other than yeast proteins may be usedfor the spacer arm; however, for any spacer arm protein, it is mostdesirable to have the immunogenic response be directed against thetarget antigen rather than the spacer arm protein. As such, if otherproteins are used for the spacer arm, then the spacer arm protein thatis used should not generate such a large immune response to the spacerarm protein itself such that the immune response to the targetantigen(s) is overwhelmed. One of skill in the art should aim for asmall immune response to the spacer arm protein relative to the immuneresponse for the target antigen(s). Spacer arms can be constructed tohave cleavage sites (e.g., protease cleavage sites) that allow theantigen to be readily removed or processed away from the yeast, ifdesired. Any known method of determining the magnitude of immuneresponses can be used (e.g., antibody production, lytic assays, etc.)and are readily known to one of skill in the art.

Another method for positioning the target antigen(s) or other proteinsto be exposed on the yeast surface is to use signal sequences such asglycosylphosphatidyl inositol (GPI) to anchor the target to the yeastcell wall. Alternatively, positioning can be accomplished by appendingsignal sequences that target the antigen(s) or other proteins ofinterest into the secretory pathway via translocation into theendoplasmic reticulum (ER) such that the antigen binds to a proteinwhich is bound to the cell wall (e.g., cwp).

In one aspect, the spacer arm protein is a yeast protein. The yeastprotein can consist of between about two and about 800 amino acids of ayeast protein. In one embodiment, the yeast protein is about 10 to 700amino acids. In another embodiment, the yeast protein is about 40 to 600amino acids. Other embodiments of the invention include the yeastprotein being at least 250 amino acids, at least 300 amino acids, atleast 350 amino acids, at least 400 amino acids, at least 450 aminoacids, at least 500 amino acids, at least 550 amino acids, at least 600amino acids, or at least 650 amino acids. In one embodiment, the yeastprotein is at least 450 amino acids in length.

Use of yeast proteins can stabilize the expression of fusion proteins inthe yeast vehicle, prevents posttranslational modification of theexpressed fusion protein, and/or targets the fusion protein to aparticular compartment in the yeast (e.g., to be expressed on the yeastcell surface). For delivery into the yeast secretory pathway, exemplaryyeast proteins to use include, but are not limited to: Aga (including,but not limited to, Aga1 and/or Aga2); SUC2 (yeast invertase); alphafactor signal leader sequence; CPY; Cwp2p for its localization andretention in the cell wall; BUD genes for localization at the yeast cellbud during the initial phase of daughter cell formation; Flo1p; Pir2p;and Pir4p.

Other sequences can be used to target, retain and/or stabilize theprotein to other parts of the yeast vehicle, for example, in the cytosolor the mitochondria. Examples of suitable yeast protein that can be usedfor any of the embodiments above include, but are not limited to, SECT;phosphoenolpyruvate carboxykinase PCK1, phosphoglycerokinase PGK andtriose phosphate isomerase TPI gene products for their repressibleexpression in glucose and cytosolic localization; the heat shockproteins SSA1, SSA3, SSA4, SSC1, whose expression is induced and whoseproteins are more thermostable upon exposure of cells to heat treatment;the mitochondrial protein CYC1 for import into mitochondria; ACT1.

In one embodiment, control of the amount of yeast glycosylation is usedto control the expression of antigens by the yeast, particularly on thesurface. The amount of yeast glycosylation can affect the immunogenicityand antigenicity of the antigen expressed on the surface, since sugarmoieties tend to be bulky. As such, the existence of sugar moieties onthe surface of yeast and its impact on the three-dimensional spacearound the target antigen(s) should be considered in the modulation ofyeast according to the invention. Any method can be used to reduce theamount of glycosylation of the yeast (or increase it, if desired). Forexample, one could use a yeast mutant strain that has been selected tohave low glycosylation (e.g. mnnl, ochl and mnn9 mutants), or one couldeliminate by mutation the glycosylation acceptor sequences on the targetantigen. Alternatively, one could use a yeast with abbreviatedglycosylation patterns, e.g. Pichia. One can also treat the yeast usingmethods that reduce or alter the glycosylation.

In one embodiment of the present invention, as an alternative toexpression of an antigen or other protein recombinantly in the yeastvehicle, a yeast vehicle is loaded intracellularly with the protein orpeptide, or with carbohydrates or other molecules that serve as anantigen and/or are useful as immunomodulatory agents or biologicalresponse modifiers according to the invention. Subsequently, the yeastvehicle, which now contains the antigen and/or other proteinsintracellularly, can be administered to the patient or loaded into acarrier such as a dendritic cell. Peptides and proteins can be inserteddirectly into yeast vehicles of the present invention by techniquesknown to those skilled in the art, such as by diffusion, activetransport, liposome fusion, electroporation, phagocytosis, freeze-thawcycles and bath sonication. Yeast vehicles that can be directly loadedwith peptides, proteins, carbohydrates, or other molecules includeintact yeast, as well as spheroplasts, ghosts or cytoplasts, which canbe loaded with antigens and other agents after production.Alternatively, intact yeast can be loaded with the antigen and/or agent,and then spheroplasts, ghosts, cytoplasts, or subcellular particles canbe prepared therefrom. Any number of antigens and/or other agents can beloaded into a yeast vehicle in this embodiment, from at least 1, 2, 3, 4or any whole integer up to hundreds or thousands of antigens and/orother agents, such as would be provided by the loading of amicroorganism or portions thereof, for example.

In another embodiment of the present invention, an antigen and/or otheragent is physically attached to the yeast vehicle. Physical attachmentof the antigen and/or other agent to the yeast vehicle can beaccomplished by any method suitable in the art, including covalent andnon-covalent association methods which include, but are not limited to,chemically crosslinking the antigen and/or other agent to the outersurface of the yeast vehicle or biologically linking the antigen and/orother agent to the outer surface of the yeast vehicle, such as by usingan antibody or other binding partner. Chemical cross-linking can beachieved, for example, by methods including glutaraldehyde linkage,photoaffinity labeling, treatment with carbodiimides, treatment withchemicals capable of linking di-sulfide bonds, and treatment with othercross-linking chemicals standard in the art. Alternatively, a chemicalcan be contacted with the yeast vehicle that alters the charge of thelipid bilayer of yeast membrane or the composition of the cell wall sothat the outer surface of the yeast is more likely to fuse or bind toantigens and/or other agent having particular charge characteristics.Targeting agents such as antibodies, binding peptides, solublereceptors, and other ligands may also be incorporated into an antigen asa fusion protein or otherwise associated with an antigen for binding ofthe antigen to the yeast vehicle.

In yet another embodiment, the yeast vehicle and the antigen or otherprotein are associated with each other by a more passive, non-specificor non-covalent binding mechanism, such as by gently mixing the yeastvehicle and the antigen or other protein together in a buffer or othersuitable formulation (e.g., admixture).

In one embodiment of the invention, the yeast vehicle and the antigen orother protein are both loaded intracellularly into a carrier such as adendritic cell or macrophage to form the therapeutic composition orvaccine of the present invention.

In one embodiment, intact yeast (with or without expression ofheterologous antigens or other proteins) can be ground up or processedin a manner to produce yeast cell wall preparations, yeast membraneparticles or yeast fragments (i.e., not intact) and the yeast fragmentscan, in some embodiments, be provided with or administered with othercompositions that include antigens (e.g., DNA vaccines, protein subunitvaccines, killed or inactivated pathogens) to enhance immune response.For example, enzymatic treatment, chemical treatment or physical force(e.g., mechanical shearing or sonication) can be used to break up theyeast into parts that are used as an adjuvant.

In one embodiment of the invention, yeast vehicles useful in theinvention include yeast vehicles that have been killed or inactivated.Killing or inactivating of yeast can be accomplished by any of a varietyof suitable methods known in the art. For example, heat inactivation ofyeast is a standard way of inactivating yeast, and one of skill in theart can monitor the structural changes of the target antigen, ifdesired, by standard methods known in the art. Alternatively, othermethods of inactivating the yeast can be used, such as chemical,electrical, radioactive or UV methods. See, for example, the methodologydisclosed in standard yeast culturing textbooks such as Methods ofEnzymology, Vol. 194, Cold Spring Harbor Publishing (1990). Any of theinactivation strategies used should take the secondary, tertiary orquaternary structure of the target antigen into consideration andpreserve such structure as to optimize its immunogenicity.

Effective conditions for the production of recombinant yeast vehiclesand expression of the antigen and/or other protein (e.g., an agent asdescribed herein) by the yeast vehicle include an effective medium inwhich a yeast strain can be cultured. An effective medium is typicallyan aqueous medium comprising assimilable carbohydrate, nitrogen andphosphate sources, as well as appropriate salts, minerals, metals andother nutrients, such as vitamins and growth factors. The medium maycomprise complex nutrients or may be a defined minimal medium. Yeaststrains of the present invention can be cultured in a variety ofcontainers, including, but not limited to, bioreactors, Erlenmeyerflasks, test tubes, microtiter dishes, and Petri plates. Culturing iscarried out at a temperature, pH and oxygen content appropriate for theyeast strain. Such culturing conditions are well within the expertise ofone of ordinary skill in the art (see, for example, Guthrie et al.(eds.), 1991, Methods in Enzymology, vol. 194, Academic Press, SanDiego).

In some aspects of the invention, the yeast are grown under neutral pHconditions, and particularly, in a media maintained at a pH level of atleast 5.5, namely the pH of the culture media is not allowed to dropbelow pH 5.5. In other aspects, the yeast is grown at a pH levelmaintained at about 5.5. One of skill in the art will appreciate thatthe culturing process includes not only the start of the yeast culturebut the maintenance of the culture as well. As yeast culturing is knownto turn acidic (i.e., lowering the pH) over time, care must be taken tomonitor the pH level during the culturing process. This process isdescribed in detail in WO 2008/097863, published 14 Aug. 2008.

Yeast vehicles can be formulated into yeast-based immunotherapycompositions or products of the present invention, includingpreparations to be administered to a subject directly or first loadedinto a carrier, using a number of techniques known to those skilled inthe art. For example, yeast vehicles can be dried by lyophilization.Formulations comprising yeast vehicles can also be prepared by packingyeast in a cake or a tablet, such as is done for yeast used in baking orbrewing operations. In addition, yeast vehicles can be mixed with apharmaceutically acceptable excipient, such as an isotonic buffer thatis tolerated by a host or host cell. Examples of such excipients includewater, saline, Ringer's solution, dextrose solution, Hank's solution,and other aqueous physiologically balanced salt solutions. Nonaqueousvehicles, such as fixed oils, sesame oil, ethyl oleate, or triglyceridesmay also be used. Other useful formulations include suspensionscontaining viscosity-enhancing agents, such as sodiumcarboxymethylcellulose, sorbitol, glycerol or dextran. Excipients canalso contain minor amounts of additives, such as substances that enhanceisotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer and Tris buffer, while examples ofpreservatives include thimerosal, m- or o-cresol, formalin and benzylalcohol. Standard formulations can either be liquid injectables orsolids which can be taken up in a suitable liquid as a suspension orsolution for injection. Thus, in a non-liquid formulation, the excipientcan comprise, for example, dextrose, human serum albumin, and/orpreservatives to which sterile water or saline can be added prior toadministration.

Antigens Useful in the Immunotherapy Compositions of the Invention

According to the present invention, the general use herein of the term“antigen” refers: to any portion of a protein (peptide, partial protein,full-length protein), wherein the protein is naturally occurring orsynthetically derived, to a cellular composition (whole cell, celllysate or disrupted cells), to an organism (whole organism, lysate ordisrupted cells) or to a carbohydrate, or other molecule, or a portionthereof. An antigen may, in some embodiments, elicit an antigen-specificimmune response (e.g., a humoral and/or a cell-mediated immune response)against the same or similar antigens that are encountered by an elementof the immune system (e.g., T cells, antibodies). The term “cancerantigen” can be used interchangeably herein with the terms“tumor-specific antigen”, “tumor-associated antigen”, “cancer-associatedtarget” or “tumor-associated target”.

An antigen can be as small as a single epitope, or larger, and caninclude multiple epitopes. As such, the size of an antigen can be assmall as about 5-12 amino acids (e.g., a peptide) and as large as: apartial protein, a full length protein, including a multimer and fusionprotein, chimeric protein, or agonist protein or peptide. In addition,antigens can include carbohydrates.

When referring to stimulation of an immune response, the term“immunogen” is a subset of the term “antigen”, and therefore, in someinstances, can be used interchangeably with the term “antigen”. Animmunogen, as used herein, describes an antigen which elicits a humoraland/or cell-mediated immune response (i.e., is immunogenic), such thatadministration of the immunogen to an individual mounts anantigen-specific immune response against the same or similar antigensthat are encountered by the immune system of the individual.

An “immunogenic domain” of a given antigen can be any portion, fragmentor epitope of an antigen (e.g., a peptide fragment or subunit or anantibody epitope or other conformational epitope) that contains at leastone epitope that acts as an immunogen when administered to an animal.For example, a single protein can contain multiple different immunogenicdomains. Immunogenic domains need not be linear sequences within aprotein, such as in the case of a humoral immune response.

An “epitope” is defined herein as a single immunogenic site within agiven antigen that is sufficient to elicit an immune response. Those ofskill in the art will recognize that T cell epitopes are different insize and composition from B cell epitopes, and that epitopes presentedthrough the Class I MHC pathway differ from epitopes presented throughthe Class II MHC pathway. Epitopes can be linear sequence orconformational epitopes (conserved binding regions).

Antigens useful in any of the immunotherapy compositions describedherein can include any antigen(s) or immunogenic domain(s) thereofagainst which it is desirable to elicit an immune response, and inparticular, include any antigen(s) or immunogenic domain(s) thereof forwhich a therapeutic immune response against such antigen would bebeneficial to an individual. The antigen can include, but is not limitedto: a cancer antigen, a viral antigen, an overexpressed mammalian cellsurface molecule, a bacterial antigen, a fungal antigen, a protozoanantigen, a helminth antigen, an ectoparasite antigen, a mammalian cellmolecule harboring one or more mutated amino acids, a protein normallyexpressed pre- or neo-natally by mammalian cells, a protein whoseexpression is induced by insertion of an epidemiologic agent (e.g.virus), a protein whose expression is induced by gene translocation, anda protein whose expression is induced by mutation of regulatorysequences.

In one aspect of the invention, antigens useful in one or moreimmunotherapy compositions of the invention include any cancer ortumor-associated antigen. In one aspect, the antigen includes an antigenassociated with a preneoplastic or hyperplastic state. The antigen mayalso be associated with, or causative of cancer. Such an antigen may bea tumor-specific antigen, a tumor-associated antigen (TAA) ortissue-specific antigen, an epitope thereof, or an epitope agonistthereof. Cancer antigens include, but are not limited to, antigens fromany tumor or cancer, including, but not limited to, melanomas, squamouscell carcinoma, breast cancers, head and neck carcinomas, thyroidcarcinomas, soft tissue sarcomas, bone sarcomas, testicular cancers,prostatic cancers, ovarian cancers, bladder cancers, skin cancers, braincancers, angiosarcomas, hemangiosarcomas, mast cell tumors, leukemias,lymphomas, primary hepatic cancers, lung cancers, pancreatic cancers,gastrointestinal cancers (including colorectal cancers), renal cellcarcinomas, hematopoietic neoplasias and metastatic cancers thereof.

Suitable cancer antigens include but are not limited to carcinoembryonicantigen (CEA) and epitopes thereof such as CAP-1, CAP-1-6D (GenBankAccession No. M29540 or Zaremba et al., 1997, Cancer Research57:4570-4577), MART-1 (Kawakami et al, J. Exp. Med. 180:347-352, 1994),MAGE-1 (U.S. Pat. No. 5,750,395), MAGE-3, GAGE (U.S. Pat. No.5,648,226), GP-100 (Kawakami et al., Proc. Nat'l Acad. Sci. USA91:6458-6462, 1992), MUC-1 (e.g., Jerome et al., J. Immunol.,151:1654-1662 (1993)), MUC-2, mutated Ras oncoprotein (see, e.g., U.S.Pat. Nos. 7,465,454 and 7,563,447), normal and mutated p53 oncoproteins(Hollstein et al Nucleic Acids Res. 22:3551-3555, 1994), PSMA (prostatespecific membrane antigen; Israeli et al., Cancer Res. 53:227-230,1993), tyrosinase (Kwon et al PNAS 84:7473-7477, 1987), TRP-1 (gp75)(Cohen et al Nucleic Acid Res. 18:2807-2808, 1990; U.S. Pat. No.5,840,839), NY-ESO-1 (Chen et al PNAS 94: 1914-1918, 1997), TRP-2(Jackson et al., EMBOJ 11:527-535, 1992), TAG72, KSA, CA-125, PSA(prostate specific antigen; Xue et al., The Prostate, 30:73-78 (1997)),HER-2/neu/c-erb/B2, (U.S. Pat. No. 5,550,214), EGFR (epidermal growthfactor receptor; Harris et al., Breast Cancer Res. Treat, 29:1-2(1994)), hTERT, p′73, B-RAF (B-Raf proto-oncogeneserine/threonine-protein kinase; Sithanandam et al., (1990), Oncogene5(12):1775-80), adenomatous polyposis coli (APC), Myc, von Hippel-Lindauprotein (VHL), Rb-1, Rb-2, androgen receptor (AR), Smad4, MDR1 (alsoknown as P-glycoprotein), Flt-3, BRCA-1 (breast cancer 1; U.S. Pat. No.5,747,282), BRCA-2 (breast cancer 2; U.S. Pat. No. 5,747,282)), Bcr-Ab1,pax3-fkhr, ews-fli-1, Brachyury (GenBank Accession Nos. NP_003172.1 orNM 003181.2; Edwards et al., 1996, Genome Res. 6:226-233), HERV-H (humanendogenous retrovirus H), HERV-K (human endogenous retrovirus K), TWIST(GenBank Accession Nos. NM_000474 and NP_000465), Mesothelin (Kojima etal., 1995, 1 Biol. Chem. 270(37):21984-90; Chang and Pastan, 1996, Proc.Natl. Acad. Sci. U.S.A. 93(1):136-40), NGEP (New Gene Expressed inProstate; Bera et al., 2004, Proc. Natl. Acad. Sci. U.S.A.101(9):3059-3064; Cereda et al., 2010, Cancer Immunol. Immunother.59(1):63-71; GenBank Accession Nos. AAT40139 or AAT40140), modificationsof such antigens and tissue specific antigens, splice variants of suchantigens, and/or epitope agonists of such antigens. Other cancerantigens are known in the art. Other cancer antigens may also beidentified, isolated and cloned by methods known in the art such asthose disclosed in U.S. Pat. No. 4,514,506. Cancer antigens may alsoinclude one or more growth factors and splice variants of each.

In one aspect of the invention, the cancer antigen is carcinoembryonicantigen (CEA), a polypeptide comprising or consisting of epitopesthereof such as CAP-1, CAP-1-6D (GenBank Accession No. M29540 or Zarembaet al., 1997, Cancer Research 57:4570-4577), a modified CEA, a splicevariant of CEA, an epitope agonist of such CEA proteins, and/or a fusionprotein comprising at least one immunogenic domain of CEA or an agonistepitope thereof. In one aspect, the CEA is a modified CEA correspondingto the modified CEA having an amino acid sequence represented by SEQ IDNO:46 in U.S. Patent Publication No. US 2007_0048860, published Mar. 1,2007, which is encoded by a nucleic acid sequence of SEQ ID NO:45. Inone aspect, the antigen is a modified CEA having an amino acid sequencerepresented by SEQ ID NO:2 herein, which is encoded by a nucleic acidsequence represented by SEQ ID NO:1.

In one aspect of the invention, the antigen is a mutated Rasoncoprotein. Exemplary Ras oncoproteins have been described, forexample, in U.S. Pat. Nos. 7,465,454 and 7,563,447. Ras is one exampleof an oncoprotein in which several mutations are known to occur atparticular positions and be associated with the development of one ormore types of cancer. Therefore, one can construct fusion proteins thatconsist of peptides containing a particular residue that is known to bemutated in certain cancers, wherein each domain contains a differentmutation at that site in order to cover several or all known mutationsat that site. A fusion protein useful in the present invention may haveone, two, or multiple domains, wherein each domain consists of a peptidefrom a particular protein (the same or different proteins), each peptideconsisting of at least 4 amino acid residues flanking either side of andincluding an epitope or mutated amino acid, such as a mutated amino acidthat is found in the protein, wherein the mutation is associated with aparticular disease (e.g., cancer). For example, with regard to Ras, onemay provide one, two, three, or more immunogenic domains comprising atleast 4 amino acids on either side of and including position 12, whereineach domain has a different substitution for the glycine that normallyoccurs in the non-mutated Ras protein (e.g., a substitution of a valine,a cysteine, an arginine, an aspartate, a serine, or an alanine, for theglycine). As another example, one may provide one, two, three, or moreimmunogenic domains comprising at least 4 amino acids on either side ofand including position 13, wherein each domain has a differentsubstitution for the glycine that normally occurs in the non-mutated Rasprotein (e.g., a substitution of an aspartate for the glycine). As yetanother example, one may provide one, two, three, or more immunogenicdomains comprising at least 4 amino acids on either side of andincluding position 61, wherein each domain has a different substitutionfor the glutamine that normally occurs in the non-mutated Ras protein(e.g., a substitution of a leucine, an arginine, or a histidine, for theglutamine). In one example, the cancer antigen comprises fragments of atleast 5-9 contiguous amino acid residues of a wild-type Ras proteincontaining amino acid positions 12, 13, 59, 61 or 76 relative to thewild-type Ras protein, wherein the amino acid residues at positions 12,13, 59, 61 or 76 are mutated with respect to the wild-type Ras protein.In one aspect, the fusion protein construct consists of at least onepeptide that is fused in frame with another mutated tumor antigen (e.g.,a Ras protein comprising at least one mutation relative to a wild-typeRas protein sequence), wherein the peptide is selected from the groupconsisting of: (a) a peptide comprising at least from positions 8-16 ofwild-type Ras (human or murine K-Ras, N-Ras or H-Ras), wherein the aminoacid residue at position 12 with respect to wild-type Ras is mutated ascompared to wild-type Ras; (b) a peptide comprising at least frompositions 9-17 of wild-type Ras, wherein the amino acid residue atposition 13 with respect to wild-type Ras is mutated as compared towild-type Ras; (c) a peptide comprising at least from positions 55-63 ofwild-type Ras, wherein the amino acid residue at position 59 withrespect to SEQ ID NO:3 is mutated as compared to wild-type Ras; (d) apeptide comprising at least from positions 57-65 of wild-type Ras,wherein the amino acid residue at position 61 with respect to wild-typeRas is mutated as compared to wild-type Ras; or (e) a peptide comprisingat least from positions 72-80 of wild-type Ras, wherein the amino acidresidue at position 76 with respect to wild-type Ras is mutated ascompared to wild-type Ras. It is noted that these positions areidentical among human and mouse K-Ras, N-Ras and H-Ras, since human andmouse sequences are identical in this region of the protein and sinceK-Ras, H-Ras and N-Ras are identical in this region. In one aspect, thea Ras fusion protein suitable for use as an antigen in the presentinvention is selected from: SEQ ID NO:4 (encoded by a nucleic acidsequence represented herein as SEQ ID NO:3), SEQ ID NO:6 (encoded by anucleic acid sequence represented herein as SEQ ID NO:5), SEQ ID NO:8(encoded by a nucleic acid sequence represented herein as SEQ ID NO:7),and/or SEQ ID NO:10 (encoded by a nucleic acid sequence representedherein as SEQ ID NO:9).

In one aspect of the invention, the antigen is human Brachyury. Theamino acid sequence for human Brachyury is represented herein by SEQ IDNO:15, which is encoded by a nucleic acid sequence represented by SEQ IDNO:14.

In another aspect of the invention, antigens useful in one or moreimmunotherapy compositions of the invention include any antigensassociated with a pathogen or a disease or condition caused by orassociated with a pathogen. Such antigens include, but are not limitedto, viral antigens, fungal antigens, bacterial antigens, helminthantigens, parasitic antigens, ectoparasite antigens, protozoan antigens,or antigens from any other infectious agent.

In one aspect, the antigen is from virus, including, but not limited to,adenoviruses, arena viruses, bunyaviruses, coronaviruses, coxsackieviruses, cytomegaloviruses, Epstein-Ban viruses, flaviviruses,hepadnaviruses, hepatitis viruses, herpes viruses, influenza viruses,lentiviruses, measles viruses, mumps viruses, myxoviruses,orthomyxoviruses, papilloma viruses, papovaviruses, parainfluenzaviruses, paramyxoviruses, parvoviruses, picornaviruses, poxviruses,rabies viruses, respiratory syncytial viruses, reoviruses,rhabdoviruses, rubella viruses, togaviruses, and varicella viruses.Other viruses include T-lymphotrophic viruses, such as human T-celllymphotrophic viruses (HTLVs, such as HTLV-I and HTLV-II), bovineleukemia viruses (BLVS) and feline leukemia viruses (FLVs). Thelentiviruses include, but are not limited to, human (HIV, includingHIV-1 or HIV-2), simian (SIV), feline (FIV) and canine (CIV)immunodeficiency viruses. In one embodiment, viral antigens includethose from non-oncogenic viruses.

In another aspect, the antigen is from an infectious agent from a genusselected from: Aspergillus, Bordatella, Brugia, Candida, Chlamydia,Coccidia, Cryptococcus, Dirofilaria, Escherichia, Francisella,Gonococcus, Histoplasma, Leishmania, Mycobacterium, Mycoplasma,Paramecium, Pertussis, Plasmodium, Pneumococcus, Pneumocystis,Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus,Toxoplasma, Vibriocholerae, and Yersinia. In one aspect, the infectiousagent is selected from Plasmodium falciparum or Plasmodium vivax.

In one aspect, the antigen is from a bacterium from a family selectedfrom: Enterobacteriaceae, Micrococcaceae, Vibrionaceae, Pasteurellaceae,Mycoplasmataceae, and Rickettsiaceae. In one aspect, the bacterium is ofa genus selected from: Pseudomonas, Bordetella, Mycobacterium, Vibrio,Bacillus, Salmonella, Francisella, Staphylococcus, Streptococcus,Escherichia, Enterococcus, Pasteurella, and Yersinia. In one aspect, thebacterium is from a species selected from: Pseudomonas aeruginosa,Pseudomonas mallei, Pseudomonas pseudomallei, Bordetella pertussis,Mycobacterium tuberculosis, Mycobacterium leprae, Francisellatularensis, Vibrio cholerae, Bacillus anthracis, Salmonella enteric,Yersinia pestis, Escherichia coli and Bordetella bronchiseptica.

In one aspect, the antigen is from a fungus, such a fungus including,but not limited to, a fungus from Saccharomyces spp., Aspergillus spp.,Cryptococcus spp., Coccidioides spp., Neurospora spp., Histoplasma spp.,or Blastomyces spp.. In one aspect, the fungus is from a speciesselected from: Aspergillus fumigatus, A. flavus, A. niger, A. terreus,A. nidulans, Coccidioides immitis, Coccidioides posadasii orCryptococcus neoformans. The most common species of Aspergillus causinginvasive disease include A. fumigatus, A. flavus, A. niger, A. terreusand A. nidulans, and may be found, for example, in patients who haveimmunosuppression or T-cell or phagocytic impairment. A. fumigatus hasbeen implicated in asthma, aspergillomas and invasive aspergillosis.Coccidioidomycosis, also known as San Joaquin Valley Fever, is a fungaldisease caused by Coccidioides immitis, and can lead to acuterespiratory infections and chronic pulmonary conditions or disseminationto the meninges, bones, and joints. Cryptococcosis-associated conditionsare also targeted by methods of the invention, for example, in anon-immunosuppressed or immunosuppressed subject, such as a subject whois infected with HIV.

In some embodiments, the antigen is a fusion protein. In one aspect ofthe invention, a fusion protein can include two or more antigens. In oneaspect, the fusion protein can include two or more immunogenic domainsand/or two or more epitopes of one or more antigens. Any combination ofantigens, immunogenic domains thereof, and/or epitopes thereof arecontemplated for use in the compositions of the invention. Animmunotherapeutic composition containing such antigens, immunogenicdomains thereof, and/or epitopes thereof may provide antigen-specificimmunization in a broad range of patients. For example, a fusion proteinuseful in the present invention may have multiple domains (two or moredomains), wherein each domain consists of a peptide or polypeptide froma particular protein, the peptide or polypeptide consisting of at least4 amino acid residues flanking either side of and including a mutatedamino acid that is found in the protein, wherein the mutation isassociated with a particular disease or condition.

In one embodiment, fusion proteins that are used as a component of theyeast-based immunotherapeutic composition useful in the invention areproduced using constructs that are particularly useful for theexpression of heterologous antigens in yeast. Typically, the desiredantigenic protein(s) or peptide(s) are fused at their amino-terminal endto: (a) a specific synthetic peptide that stabilizes the expression ofthe fusion protein in the yeast vehicle or prevents posttranslationalmodification of the expressed fusion protein (such peptides aredescribed in detail, for example, in U.S. Patent Publication No.2004-0156858 A1, published Aug. 12, 2004, incorporated herein byreference in its entirety); (b) at least a portion of an endogenousyeast protein, wherein either fusion partner provides significantlyenhanced stability of expression of the protein in the yeast and/or aprevents post-translational modification of the proteins by the yeastcells (such proteins are also described in detail, for example, in U.S.Patent Publication No. 2004-0156858 A1, supra); and/or (c) at least aportion of a yeast protein that causes the fusion protein to beexpressed on the surface of the yeast (e.g., an Aga protein, asdescribed in detail in WO 2008/019366). In addition, the presentinvention includes the use of peptides that are fused to the C-terminusof the antigen-encoding construct, particularly for use in the selectionand identification of the protein. Such peptides include, but are notlimited to, any synthetic or natural peptide, such as a peptide tag(e.g., 6×His) or any other short epitope tag. Peptides attached to theC-terminus of an antigen according to the invention can be used with orwithout the addition of the N-terminal peptides discussed above.

According to the invention, in one embodiment, the two or more differentimmunotherapy compositions preferably target the same antigen(s). Inthis embodiment, the same antigen(s) or immunogenic domain(s) thereofare typically expressed by each of the immunotherapy compositionvectors, although the antigen(s) and/or immunogenic domain(s) thereofmay be admixed with one or both of the compositions, and/or in the caseof the yeast-based immunotherapy composition, the antigen may beattached to the yeast vehicle and/or carried inside the yeast vehicle.In one embodiment, both compositions may target the same antigen(s),although each composition may target a different epitope(s) orimmunogenic domain(s) within the same antigen. In one embodiment, eachimmunotherapy compositions targets a different antigen(s) and/orimmunogenic domain and/or epitope thereof. For example, it may beadvantageous to target one antigen, such as Ras, using one compositionand another cancer antigen that is also expressed in the same or asubset of the same cancers, such as CEA, using the other immunotherapycomposition. In another embodiment, a combination of one type ofimmunotherapy composition is provided, wherein there are at least twodifferent antigen compositions within the same type (e.g., a combinationof a yeast-based immunotherapy composition expressing mutated Ras or afusion protein comprising multiple immunogenic domains of mutated Ras,and a yeast-based immunotherapy composition expressing CEA or a modifiedCEA as described herein). This combination is administered concurrentlywith the other type of immunotherapy composition (e.g., a virus-basedcomposition) that expresses the same or different antigen(s). In oneaspect of such an embodiment, the various combinations can be mixed orin another aspect, need not be physically mixed, but can rather beadministered concurrently such as to the same site or different sites,or within the same administration period. In yet another embodiment, oneof the two or more different immunotherapy compositions targets one ormore antigens, and the other of the immunotherapy compositions isprovided as an adjuvant, without necessarily targeting any antigen(i.e., the composition is used for its non-antigen-specificimmunotherapy benefits), or without necessarily targeting the samecancer antigen as the other composition. More particularly, because eachof the immunotherapy vectors described herein has been show tocontribute vector-specific effects to the efficacy of the composition,in some embodiments, only one vector may be associated with or expressthe antigen, and the other vector is used concurrently to contributenon-antigen-specific immunotherapy effects.

Methods of Use of the Compositions of the Invention

In the methods of the present invention, two or more immunotherapycompositions as described herein are first administered concurrently toan individual. As used herein with respect to administration of acomposition, the term “concurrently” means to administer each of thecompositions, and particularly, the first dose of such compositions,essentially at the same time or within the same dosing period, or withina time period during which the initial effects of priming of the immunesystem by the immunotherapy compositions occur (e.g., within 1-2 daysand preferably less). For clarity, concurrent administration does notrequire administration of all of the compositions at precisely the samemoment, but rather, the administration of all compositions should occurwithin one scheduled dosing of the patient in order to prime the immunesystem with each of the compositions concurrently (e.g., one compositionmay be administered first, followed immediately or closely by theadministration of the second composition, and so on). In somecircumstances, such as when the compositions are administered to thesame site, the compositions may be provided in admixture, although evenwhen administered at the same site, sequential administration of eachcomposition during the same dosing period may be preferred. In oneaspect, the compositions are administered within the same 1-2 days, andin one aspect, on the same day, and in one aspect, within the same 12hour period, and in one aspect, within the same 8 hour period, and inone aspect, within the same 4 hour period, and in one aspect, within thesame 1, 2 or 3 hour period, and in one aspect, within the same 1, 2, 3,4, 6, 7, 8, 9, or 10 minutes.

In some circumstances, either the first or the second immunotherapycomposition is administered more frequently than the other. For example,in one aspect, when the first immunotherapy composition is a virus-basedimmunotherapy composition, and the second immunotherapy composition is ayeast-based immunotherapy composition, the second immunotherapycomposition may be administered more frequently than the firstimmunotherapy composition. For example, in one aspect, in betweenconcurrent administrations of the first and second immunotherapycomposition, the second immunotherapy composition may be administeredone, two, three or more additional times. For instance, because theimmunization with the virus-based immunotherapy composition results inextended presentation of antigen, it may not be necessary or beneficialto administer this composition on shorter frequencies, whereasyeast-based immunotherapy compositions present discrete bolus' ofantigen, and so they can be administered more frequently without fearsof inhibiting the immune response. For example, in one aspect, thevirus-based immunotherapy composition is administered every 2, 3 or 4 ormore weeks, while yeast-based immunotherapy compositions areadministered at 1 week intervals, which may be extended to longerintervals (2, 3 or 4 weeks or more) as the total period of therapyincreases.

In one embodiment of the invention, the two or more immunotherapycompositions are administered concurrently, but to different physicalsites in the patient. For example, one composition can be administeredto a site on one side of the individual's body and the other compositioncan be administered to a site on the other side of the individual'sbody. As another example, one composition can be administered at a sitenear a particular draining lymph node, and the other composition can beadministered at a site near a different draining lymph node. In anotherembodiment, the two or more different immunotherapy compositions areadministered concurrently and to the same or substantially adjacentsites in the patient. A substantially adjacent site is a site that isnot precisely the same injection site to which the first composition isadministered, but that is in close proximity (is next to or near to) thefirst injection site. In one embodiment, the two or more differentimmunotherapy compositions are administered in admixture. In one aspectof the invention, a virus-based immunotherapy composition isadministered subcutaneously, intramuscularly, or intratumorally, and ayeast-based composition of the invention is administered subcutaneously.

Some embodiments may include combinations of administration approaches.For example, using the exemplary case of concurrent administration of ayeast-based immunotherapy composition (a first composition) and avirus-based immunotherapy composition (a second composition), oneportion of the dose of the yeast-based composition (e.g., one fractionof the total dose of yeast-based composition) may be administered inadmixture with or to the same or adjacent site as a portion of or all ofthe dose of the virus-based immunotherapy composition, and then theremaining portion(s) of the dose of the yeast-based immunotherapycomposition are administered to other site(s) in the individual.Similarly, a portion of the dose of the virus-based composition can beadministered in admixture with or to the same or adjacent site as aportion of or all of the dose of the yeast-based composition, and thenthe remaining portion(s) of the virus-based immunotherapy compositionare administered to other site(s) in the individual. In one embodiment,within a single dose amount of virus-based and/or yeast-basedimmunotherapy composition that is to be administered in portions todifferent sites on the individual, some portions may contain or expressthe target antigen and others may not (i.e., the others may be emptyvectors or encode a costimulatory molecule, cytokine, or othernon-antigen agent).

Administration of a vaccine or composition can be systemic, mucosaland/or proximal to the location of the target site (e.g., near a tumor).The preferred routes of administration will be apparent to those ofskill in the art, depending on the type of condition to be prevented ortreated, the antigen used, and/or the target cell population or tissue.Various acceptable methods of administration include, but are notlimited to, intravenous administration, intraperitoneal administration,intramuscular administration, intranodal administration, intracoronaryadministration, intraarterial administration (e.g., into a carotidartery), subcutaneous administration, transdermal delivery,intratracheal administration, subcutaneous administration,intraarticular administration, intraventricular administration,inhalation (e.g., aerosol), intracranial, intraspinal, intraocular,aural, intranasal, oral, pulmonary administration, impregnation of acatheter, and direct injection into a tissue. In one aspect, routes ofadministration include: intravenous, intraperitoneal, subcutaneous,intradermal, intranodal, intramuscular, transdermal, inhaled,intranasal, oral, intraocular, intraarticular, intracranial, andintraspinal. Parenteral delivery can include intradermal, intramuscular,intraperitoneal, intrapleural, intrapulmonary, intravenous,subcutaneous, atrial catheter and venal catheter routes. Aural deliverycan include ear drops, intranasal delivery can include nose drops orintranasal injection, and intraocular delivery can include eye drops.Aerosol (inhalation) delivery can also be performed using methodsstandard in the art (see, for example, Stribling et al., Proc. Natl.Acad. Sci. USA 189:11277-11281, 1992, which is incorporated herein byreference in its entirety). Other routes of administration that modulatemucosal immunity are useful in the treatment of viral infections. Suchroutes include bronchial, intradermal, intramuscular, intranasal, otherinhalatory, rectal, subcutaneous, topical, transdermal, vaginal andurethral routes. In one aspect, an immunotherapeutic composition of theinvention is administered subcutaneously. Preferred methods ofadministration include, but are not limited to, intravenous,intraperitoneal, subcutaneous, intradermal, intranodal, intramuscular,transdermal or intratumoral. The dose is administered at least once.Subsequent doses may be administered as indicated, and are typicallyutilized.

More particularly, in one embodiment, the initial concurrentadministration of the two or more different immunotherapy compositionsof the invention may be followed by subsequent booster doses of one andin one embodiment, both or all, immunotherapy compositions. Boosterdoses (boosts) may be administered any suitable period apart, and aretypically administered 1, 2, 3, 4, 5, 6, 7, or 8 weeks apart. Thebooster doses of the two or more immunotherapy compositions may beadministered concurrently or separately, as desired, but are mosttypically administered concurrently, as with the priming dose. As withthe priming dose, the method of administration can use any combinationof sites and administration strategies as described above for thepriming dose, but is not limited to the same administration protocol asfor the priming dose. For example, if the priming doses wereadministered to two different sites (one immunotherapy composition ateach site), the boosting doses can be administered to the same twosites, to a single site or adjacent sites, or to two different sites, orusing the portioning strategy described above.

In addition, the booster doses need not be formulated in exactly thesame way as the priming doses. For example, if the each of theimmunotherapy compositions in the priming dose provided an antigen(s)and/or immunogenic domain(s) thereof, in the booster dose, bothimmunotherapy compositions may again provide the antigen(s) and/orimmunogenic domain(s) thereof, or only one of the immunotherapycompositions may provide the antigen(s) and/or immunogenic domain(s)thereof, and the other immunotherapy composition may be an empty vectoror provide non-antigen agents (e.g., immunostimulatory molecules orother agents). Other possible modification of the compositions asdescribed herein is also contemplated during the boosting stage,including modifications or changes of the viral vector used (e.g., usingvaccinia virus in a priming composition and fowlpox virus in a boostingcomposition, or vice versa), modifications or changes in a yeast vehicle(e.g., change in yeast strain, change in yeast production method, changein how the yeast provides the antigen, such as expressed versus inadmixture), dose amounts, antigens or domains provided by one or more ofthe compositions, and inclusion or elimination or substitution ofimmunostimulatory molecules or other agents.

The term “unit dose” as it pertains to the inoculum of a composition ofthe invention refers to physically discrete units suitable as unitarydosages for mammals, each unit containing a predetermined quantity ofimmunotherapy composition calculated to produce the desired immunogeniceffect in association with the required diluent. The specifications forthe novel unit dose of an inoculum of this invention are dictated by andare dependent upon the unique characteristics of the particularimmunotherapy composition and the particular immunologic effect to beachieved. In providing an individual with an immunotherapy compositionof the present invention, preferably a human, the dosage of administeredrecombinant vector will vary depending upon such factors as theindividual's age, weight, height, sex, general medical condition,previous medical history, disease progression, tumor burden, pathogenburden and the like.

The inoculum is typically prepared as a solution in tolerable(acceptable) diluent such as saline, phosphate-buffered saline or otherphysiologically tolerable diluent and the like to form an aqueouspharmaceutical composition.

With respect to the recombinant virus-based immunotherapy compositionsof the invention, in general, it is desirable to provide the recipientwith a dosage of recombinant virus in the range of about 10⁵ to about10¹⁰ plaque forming units, although a lower or higher dose may beadministered, including, but not limited to, 10², 10³, 10⁴, 10⁵, 10⁶,10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹ or more plaque forming units. Examples ofmethods for administering the recombinant viral vector into individualsinclude, but are not limited to, exposure of tumor cells to therecombinant virus ex vivo, or injection of the recombinant vector intothe affected host by intravenous, subcutaneous (S.C.), intradermal(I.D.) or intramuscular (I.M.) administration of the virus.Alternatively the recombinant viral vector or combination of recombinantviral vectors may be administered locally by direct injection into acancerous lesion or tumor or topical application in a pharmaceuticallyacceptable carrier. The quantity of recombinant vector carrying thenucleic acid sequence of one or more antigens in combination withnucleic acid sequences encoding multiple costimulatory molecules to beadministered is based on the titer of virus particles. A preferred rangeof the antigen to be administered is 10⁵ to 10¹⁰ virus particles permammal, preferably a human, although a lower or higher dose may beadministered, including, but not limited to, 10², 10³, 10⁴, 10⁵, 10⁶,10⁷, 10⁸, 10⁹ 10¹⁰, 10¹¹ or more plaque forming units.

With respect to the yeast-based immunotherapy compositions of theinvention, in general, a suitable single dose is a dose that is capableof effectively providing a yeast vehicle and an antigen (if included) toa given cell type, tissue, or region of the patient body in an amounteffective to elicit an antigen-specific immune response, whenadministered one or more times over a suitable time period. For example,in one embodiment, a single dose of a yeast vehicle of the presentinvention is from about 1×10⁵ to about 5×10′ yeast cell equivalents perkilogram body weight of the organism being administered the composition.More preferably, a single dose of a yeast vehicle of the presentinvention is from about 0.1 Y.U. (1×10⁶ cells) to about 100 Y.U. (1×10⁹cells) per dose (i.e., per organism), including any interim dose, inincrements of 0.1×10⁶ cells (i.e., 1.1×10⁶, 1.2×10⁶, 1.3×10⁶ . . . ).Preferred doses include doses between 1 Y.U. and 40 Y.U. and morepreferably, between 10 Y.U. and 40 Y.U. In one embodiment, the doses areadministered at different sites on the individual but during the samedosing period. For example, a 40 Y.U. dose may be administered via byinjecting 10 Y.U. doses to four different sites on the individual duringone dosing period.

If the mammal to be immunized is already afflicted with a disease (e.g.,cancer or metastatic cancer or a chronic pathogen infection), thevaccine can be administered in conjunction with other therapeutictreatments used to treat the disease (e.g., chemotherapy, radiationtherapy, small molecule therapy, cytokine therapy, anti-viral therapy,biological response modifier therapy, surgery, etc.), in addition to theconcurrent administration of the different immunotherapy compositionsdescribed herein.

The method of use of the immunotherapy compositions of the presentinvention elicits an immune response in an individual such that theindividual is protected from the disease or condition, or from symptomsresulting from the disease or condition. As used herein, the phrase“protected from a disease” refers to preventing a disease, preventing atleast one symptom of the disease, delaying onset of a disease, reducingone or more symptoms of the disease, reducing the occurrence of thedisease, and/or reducing the severity of the disease. With respect tocancer, concurrent administration of the immunotherapy compositions ofthe invention preferably results in one or more of: prevention of tumorgrowth, delay to onset of disease, reduction of tumor burden, reductionof tumor growth, increased survival, improved organ function, and/orimproved general health of the individual. With respect to infectiousdisease and other diseases, concurrent administration of theimmunotherapy compositions preferably results in one or more of:prevention of the disease or condition, prevention of infection, delayto onset of disease, increased survival, reduction of pathogen burden(e.g., reduction of viral titer), reduction in at least one symptomresulting from the infection in the individual, reduction of organ orsystem damage resulting from the infection or disease, and improvementin organ or system function.

In the method of treatment of the present invention, the administrationof the immunotherapy compositions of the invention may be either“prophylactic” or “therapeutic”. When provided prophylactically, theimmunotherapy compositions of the present invention are provided inadvance of any symptom of a disease or condition. The prophylacticadministration of the immunotherapy compositions serves to prevent orameliorate or delay time to onset of any subsequent disease. Whenprovided therapeutically, the immunotherapy compositions are provided ator after the onset of a symptom of disease. The term, “disease” refersto any deviation from the normal health of an animal and includes astate when disease symptoms are present, as well as conditions in whicha deviation (e.g., tumor growth, infection, etc.) has occurred, butsymptoms are not yet manifested.

In any embodiment of the invention, in addition to administration of theimmunotherapy compositions of the invention to an individual, additionalexogenous immunomodulators or immunostimulatory molecules,chemotherapeutic drugs, antibiotics, antifungal drugs, antiviral drugs,cancer therapies, cytokines, and other therapeutic agents, therapeuticcompositions, or therapeutic protocols, alone or in combination, may beadministered, depending on the condition to be treated. Suitablebiological response modifiers that may be used in conjunction with theimmunotherapy compositions of the invention include, but are not limitedto, cytokines, chemokines, hormones, lipidic derivatives, peptides,proteins, polysaccharides, small molecule drugs, antibodies and antigenbinding fragments thereof (including, but not limited to, anti-cytokineantibodies, anti-cytokine receptor antibodies, anti-chemokineantibodies), vitamins, polynucleotides, nucleic acid binding moieties,aptamers, and growth modulators. Examples of exogenously added agentsand biological response modifiers include, but are not limited to,Flt-3L, cyclophosphamide, cisplatinum, gancyclovir, amphotericin B, 5fluorouracil, interleukin 2 (IL-2), interleukin 4 (IL-4), IL-6,interleukin 10 (IL-10), interleukin 12 (IL-12), type I interferon(including IFN-α) or agonists or antagonists of type I interferon or areceptor thereof; type II interferon (including IFN-γ) or agonists orantagonists of type II interferon or a receptor thereof; type IIIinterferon (including IFN-λ) or agonists or antagonists of type IIIinterferon or a receptor thereof; tumor necrosis factor-α (TNF-α);transforming growth factor-0 (TGF-β); anti-CD40; CD40L; anti-CTLA-4antibody (e.g., to release anergic T cells); T cell co-stimulators(e.g., anti-CD137, anti-CD28, anti-CD40); alemtuzumab (e.g., CAMPATH®),denileukin diftitox (e.g., ONTAK®); anti-CD4; anti-CD25; anti-PD-1,anti-PD-L1, anti-PD-L2; agents that block FOXP3 (e.g., to abrogate theactivity/kill CD4+/CD25+ T regulatory cells); Flt3 ligand, imiquimod(ALDARA™), granulocyte-macrophage colony stimulating factor (GM-CSF);granulocyte-colony stimulating factor (G-CSF), sargramostim (LEUKINE®);hormones including without limitation prolactin and growth hormone;Toll-like receptor (TLR) agonists, including but not limited to TLR-2agonists, TLR-4 agonists, TLR-7 agonists, and TLR-9 agonists; TLRantagonists, including but not limited to TLR-2 antagonists, TLR-4antagonists, TLR-7 antagonists, and TLR-9 antagonists; anti-inflammatoryagents and immunomodulators, including but not limited to, COX-2inhibitors (e.g., Celecoxib, NSAIDS), glucocorticoids, statins, andthalidomide and analogues thereof including IMIDTMs (which arestructural and functional analogues of thalidomide (e.g., REVLIMID®(lenalidomide), ACTIMID® (pomalidomide)); proinflammatory agents, suchas fungal or bacterial components or any proinflammatory cytokine orchemokine; immunotherapeutic vaccines including, but not limited to,virus-based vaccines, bacteria-based vaccines, or antibody-basedvaccines; and any other immunomodulators, immunopotentiators,anti-inflammatory agents, and/or pro-inflammatory agents.

These exogenous agents and therapies may be administered concurrentlywith the immunotherapy compositions of the invention, or at differenttime points. For example, when given to an individual in conjunctionwith chemotherapy, it may be desirable to administer the immunotherapycompositions during the “holiday” between chemotherapeutic doses, inorder to maximize the efficacy of the immunotherapy compositions.

Compositions and therapeutic vaccines of the invention can furtherinclude any other compounds that are useful for protecting a subjectfrom a particular disease or condition, including an infection by apathogen, any compounds that treat or ameliorate any symptom of such aninfection, and any compounds or treatments for cancer.

In the method of the present invention, compositions and therapeuticcompositions can be administered to animal, including any vertebrate,and particularly to any member of the Vertebrate class, Mammalia,including, without limitation, primates, rodents, livestock and domesticpets. Livestock include mammals to be consumed or that produce usefulproducts (e.g., sheep for wool production). Mammals to protect includehumans, dogs, cats, mice, rats, goats, sheep, cattle, horses and pigs.

General Techniques Useful in the Invention

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry,nucleic acid chemistry, and immunology, which are well known to thoseskilled in the art. Such techniques are explained fully in theliterature, such as, Methods of Enzymology, Vol. 194, Guthrie et al.,eds., Cold Spring Harbor Laboratory Press (1990); Biology and activitiesof yeasts, Skinner, et al., eds., Academic Press (1980); Methods inyeast genetics: a laboratory course manual, Rose et al., Cold SpringHarbor Laboratory Press (1990); The Yeast Saccharomyces: Cell Cycle andCell Biology, Pringle et al., eds., Cold Spring Harbor Laboratory Press(1997); The Yeast Saccharomyces: Gene Expression, Jones et al., eds.,Cold Spring Harbor Laboratory Press (1993); The Yeast Saccharomyces:Genome Dynamics, Protein Synthesis, and Energetics, Broach et al., eds.,Cold Spring Harbor Laboratory Press (1992); Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989) and MolecularCloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001),(jointly referred to herein as “Sambrook”); Current Protocols inMolecular Biology (F. M. Ausubel et al., eds., 1987, includingsupplements through 2001); PCR: The Polymerase Chain Reaction, (Mulliset al., eds., 1994); Harlow and Lane (1988) Antibodies, A LaboratoryManual, Cold Spring Harbor Publications, New York; Harlow and Lane(1999) Using Antibodies: A Laboratory Manual Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (jointly referred to hereinas “Harlow and Lane”), Beaucage et al. eds., Current Protocols inNucleic Acid Chemistry John Wiley & Sons, Inc., New York, 2000);Casarett and Doull's Toxicology The Basic Science of Poisons, C.Klaassen, ed., 6th edition (2001), and Vaccines, S. Plotkin and W.Orenstein, eds., 3rd edition (1999).

General Definitions

An “immunotherapeutic composition” is a composition that elicits animmune response sufficient to achieve at least one therapeutic benefitin a subject.

In general, the term “biologically active” indicates that a compound hasat least one detectable activity that has an effect on the metabolic orother processes of a cell or organism, as measured or observed in vivo(i.e., in a natural physiological environment) or in vitro (i.e., underlaboratory conditions). Accordingly, a biologically active portion orfragment or domain of a protein, for example, refers to a portion,fragment or domain that is of sufficient size to have a biologicalactivity of the full-length protein. Such activity is an activity thatis particular to that protein, rather than an activity of all proteinsas a class.

An “individual” or a “subject” or a “patient”, which terms may be usedinterchangeably, is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, farm animals, sportanimals, pets, primates, mice and rats.

According to the present invention, the term “modulate” can be usedinterchangeably with “regulate” and refers generally to upregulation ordownregulation of a particular activity. As used herein, the term“upregulate” can be used generally to describe any of: elicitation,initiation, increasing, augmenting, boosting, improving, enhancing,amplifying, promoting, or providing, with respect to a particularactivity. Similarly, the term “downregulate” can be used generally todescribe any of: decreasing, reducing, inhibiting, ameliorating,diminishing, lessening, blocking, or preventing, with respect to aparticular activity.

Reference to an isolated protein or polypeptide in the present inventionincludes full-length proteins, fusion proteins, or any fragment, domain,conformational epitope, or homologue of such proteins. Morespecifically, an isolated protein, according to the present invention,is a protein (including a polypeptide or peptide) that has been removedfrom its natural milieu (i.e., that has been subject to humanmanipulation) and can include purified proteins, partially purifiedproteins, recombinantly produced proteins, and synthetically producedproteins, for example. As such, “isolated” does not reflect the extentto which the protein has been purified. Preferably, an isolated proteinof the present invention is produced recombinantly. According to thepresent invention, the terms “modification” and “mutation” can be usedinterchangeably, particularly with regard to the modifications/mutationsto the amino acid sequence of proteins or portions thereof (or nucleicacid sequences) described herein.

As used herein, the term “homologue” is used to refer to a protein orpeptide which differs from a naturally occurring protein or peptide(i.e., the “prototype” or “wild-type” protein) by minor modifications tothe naturally occurring protein or peptide, but which maintains thebasic protein and side chain structure of the naturally occurring form.Such changes include, but are not limited to: changes in one or a fewamino acid side chains; changes one or a few amino acids, includingdeletions (e.g., a truncated version of the protein or peptide)insertions and/or substitutions; changes in stereochemistry of one or afew atoms; and/or minor derivatizations, including but not limited to:methylation, glycosylation, phosphorylation, acetylation,myristoylation, prenylation, palmitation, amidation and/or addition ofglycosylphosphatidyl inositol. A homologue can have enhanced, decreased,or substantially similar properties as compared to the naturallyoccurring protein or peptide. A homologue can include an agonist of aprotein or an antagonist of a protein. Homologues can be produced usingtechniques known in the art for the production of proteins including,but not limited to, direct modifications to the isolated, naturallyoccurring protein, direct protein synthesis, or modifications to thenucleic acid sequence encoding the protein using, for example, classicor recombinant DNA techniques to effect random or targeted mutagenesis.

A homologue of a given protein may comprise, consist essentially of, orconsist of, an amino acid sequence that is at least about 45%, or atleast about 50%, or at least about 55%, or at least about 60%, or atleast about 65%, or at least about 70%, or at least about 75%, or atleast about 80%, or at least about 85%, or at least about 90%, or atleast about 95% identical, or at least about 95% identical, or at leastabout 96% identical, or at least about 97% identical, or at least about98% identical, or at least about 99% identical (or any percent identitybetween 45% and 99%, in whole integer increments), to the amino acidsequence of the reference protein. In one embodiment, the homologuecomprises, consists essentially of, or consists of, an amino acidsequence that is less than 100% identical, less than about 99%identical, less than about 98% identical, less than about 97% identical,less than about 96% identical, less than about 95% identical, and so on,in increments of 1%, to less than about 70% identical to the naturallyoccurring amino acid sequence of the reference protein.

As used herein, unless otherwise specified, reference to a percent (%)identity refers to an evaluation of homology which is performed using:(1) a BLAST 2.0 Basic BLAST homology search using blastp for amino acidsearches and blastn for nucleic acid searches with standard defaultparameters, wherein the query sequence is filtered for low complexityregions by default (described in Altschul, S. F., Madden, T. L.,Schääffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J.(1997) “Gapped BLAST and PSI-BLAST: a new generation of protein databasesearch programs.” Nucleic Acids Res. 25:3389-3402, incorporated hereinby reference in its entirety); (2) a BLAST 2 alignment (using theparameters described below); (3) and/or PSI-BLAST with the standarddefault parameters (Position-Specific Iterated BLAST. It is noted thatdue to some differences in the standard parameters between BLAST 2.0Basic BLAST and BLAST 2, two specific sequences might be recognized ashaving significant homology using the BLAST 2 program, whereas a searchperformed in BLAST 2.0 Basic BLAST using one of the sequences as thequery sequence may not identify the second sequence in the top matches.In addition, PSI-BLAST provides an automated, easy-to-use version of a“profile” search, which is a sensitive way to look for sequencehomologues. The program first performs a gapped BLAST database search.The PSI-BLAST program uses the information from any significantalignments returned to construct a position-specific score matrix, whichreplaces the query sequence for the next round of database searching.Therefore, it is to be understood that percent identity can bedetermined by using any one of these programs.

Two specific sequences can be aligned to one another using BLAST 2sequence as described in Tatusova and Madden, (1999), “Blast 2sequences—a new tool for comparing protein and nucleotide sequences”,FEMS Microbiol Lett. 174:247-250, incorporated herein by reference inits entirety. BLAST 2 sequence alignment is performed in blastp orblastn using the BLAST 2.0 algorithm to perform a Gapped BLAST search(BLAST 2.0) between the two sequences allowing for the introduction ofgaps (deletions and insertions) in the resulting alignment. For purposesof clarity herein, a BLAST 2 sequence alignment is performed using thestandard default parameters as follows.

For blastn, using 0 BLOSUM62 matrix:Reward for match=1Penalty for mismatch=−2Open gap (5) and extension gap (2) penaltiesgap x_dropoff (50) expect (10) word size (11) filter (on)For blastp, using 0 BLOSUM62 matrix:Open gap (11) and extension gap (1) penaltiesgap x_dropoff (50) expect (10) word size (3) filter (on).

As used herein, an “agonist” is any compound or agent, including withoutlimitation small molecules, proteins, peptides, antibodies, nucleic acidbinding agents, etc., that binds to a receptor or ligand or interactswith another molecule or within a chemical or biological system andproduces or triggers a response, which may include agents that mimic theaction of a naturally occurring substance (e.g., an agonist of a proteinor peptide). An “antagonist” is any compound or agent, including withoutlimitation small molecules, proteins, peptides, antibodies, nucleic acidbinding agents, etc., that blocks or inhibits or reduces the action ofan agonist or a naturally occurring substance.

An isolated nucleic acid molecule is a nucleic acid molecule that hasbeen removed from its natural milieu (i.e., that has been subject tohuman manipulation), its natural milieu being the genome or chromosomein which the nucleic acid molecule is found in nature. As such,“isolated” does not necessarily reflect the extent to which the nucleicacid molecule has been purified, but indicates that the molecule doesnot include an entire genome or an entire chromosome in which thenucleic acid molecule is found in nature. An isolated nucleic acidmolecule can include a gene. An isolated nucleic acid molecule thatincludes a gene is not a fragment of a chromosome that includes suchgene, but rather includes the coding region and regulatory regionsassociated with the gene, but no additional genes that are naturallyfound on the same chromosome. An isolated nucleic acid molecule can alsoinclude a specified nucleic acid sequence flanked by (i.e., at the 5′and/or the 3′ end of the sequence) additional nucleic acids that do notnormally flank the specified nucleic acid sequence in nature (i.e.,heterologous sequences). Isolated nucleic acid molecule can include DNA,RNA (e.g., mRNA), or derivatives of either DNA or RNA (e.g., cDNA).Although the phrase “nucleic acid molecule” primarily refers to thephysical nucleic acid molecule and the phrase “nucleic acid sequence”primarily refers to the sequence of nucleotides on the nucleic acidmolecule, the two phrases can be used interchangeably, especially withrespect to a nucleic acid molecule, or a nucleic acid sequence, beingcapable of encoding a protein or domain of a protein. The term“polynucleotide” can also be used interchangeably with the terms“nucleic acid molecule” or “nucleic acid sequence”.

A recombinant nucleic acid molecule is a molecule that can include atleast one of any nucleic acid sequence encoding any one or more proteinsdescribed herein operatively linked to at least one of any transcriptioncontrol sequence capable of effectively regulating expression of thenucleic acid molecule(s) in the cell to be transfected. Although thephrase “nucleic acid molecule” primarily refers to the physical nucleicacid molecule and the phrase “nucleic acid sequence” primarily refers tothe sequence of nucleotides on the nucleic acid molecule, the twophrases can be used interchangeably, especially with respect to anucleic acid molecule, or a nucleic acid sequence, being capable ofencoding a protein. In addition, the phrase “recombinant molecule”primarily refers to a nucleic acid molecule operatively linked to atranscription control sequence, but can be used interchangeably with thephrase “nucleic acid molecule” which is administered to an animal.

A recombinant nucleic acid molecule includes a recombinant vector, whichis any nucleic acid sequence, typically a heterologous sequence, whichis operatively linked to the isolated nucleic acid molecule encoding afusion protein of the present invention, which is capable of enablingrecombinant production of the fusion protein, and which is capable ofdelivering the nucleic acid molecule into a host cell according to thepresent invention. Such a vector can contain nucleic acid sequences thatare not naturally found adjacent to the isolated nucleic acid moleculesto be inserted into the vector. The vector can be either RNA or DNA,either prokaryotic or eukaryotic, and preferably in the presentinvention, is a virus or a plasmid. Recombinant vectors can be used inthe cloning, sequencing, and/or otherwise manipulating of nucleic acidmolecules, and can be used in delivery of such molecules (e.g., as in aDNA composition or a viral vector-based composition). Recombinantvectors are preferably used in the expression of nucleic acid molecules,and can also be referred to as expression vectors. Preferred recombinantvectors are capable of being expressed in a transfected host cell.

In a recombinant molecule of the present invention, nucleic acidmolecules are operatively linked to expression vectors containingregulatory sequences such as transcription control sequences,translation control sequences, origins of replication, and otherregulatory sequences that are compatible with the host cell and thatcontrol the expression of nucleic acid molecules of the presentinvention. In particular, recombinant molecules of the present inventioninclude nucleic acid molecules that are operatively linked to one ormore expression control sequences. The phrase “operatively linked”refers to linking a nucleic acid molecule to an expression controlsequence in a manner such that the molecule is expressed whentransfected (i.e., transformed, transduced or transfected) into a hostcell.

According to the present invention, the term “transfection” is used torefer to any method by which an exogenous nucleic acid molecule (i.e., arecombinant nucleic acid molecule) can be inserted into a cell. The term“transformation” can be used interchangeably with the term“transfection” when such term is used to refer to the introduction ofnucleic acid molecules into microbial cells, such as algae, bacteria andyeast. In microbial systems, the term “transformation” is used todescribe an inherited change due to the acquisition of exogenous nucleicacids by the microorganism and is essentially synonymous with the term“transfection.” Therefore, transfection techniques include, but are notlimited to, transformation, chemical treatment of cells, particlebombardment, electroporation, microinjection, lipofection, adsorption,infection and protoplast fusion.

The following experimental results are provided for purposes ofillustration and are not intended to limit the scope of the invention.

EXAMPLES

The following Materials and Methods were used in the Examples below.

Mice and Tumor Cell Lines

For in vitro stimulation of lymphocytes, female C57BL/6 (H-2b) mice wereobtained from the National Cancer Institute, Frederick Cancer Researchand Development Facility (Frederick, Md.). A breeding pair of C57BL/6mice homozygous for expression of the human CEA gene (CEA-Tg) wasgenerously provided by Dr. John Shively (City of Hope, Duarte, Calif.).Homozygosity for CEA expression was confirmed by PCR analysis ofmouse-tail DNA (Greiner et al., Cancer Res 2002 Dec. 1; 62(23):6944-51).Six- to 8-week-old female mice were used for all experiments, and werehoused in micro-isolator cages under pathogen-free conditions inaccordance with AAALAC guidelines. Experimental studies were carried outunder approval of the NIH Intramural Animal Care and Use Committee. Thetarget tumor cell line EL-4 (H-2b, thymoma) was obtained from AmericanType Culture Collection (Manassas, Va.). LL/2 6 murine lungadenocarcinoma tumor cells were the gift of Dr. Chandan Guha (AlbertEinstein College of Medicine, New York, N.Y.). LL/2 murine lungcarcinoma cells expressing human CEA (LL2-CEA) were generated byretroviral transduction with CEA cDNA, as previously described (Robbinset al., Cancer Res 1991 Jul. 15; 51(14):3657-62). Cells were maintainedin complete medium (DMEM supplemented with 10% fetal bovine serum, 2 mMglutamine, 100 units/ml penicillin, and 100 m/ml streptomycin).

Vaccine Constructs

Recombinant vaccinia (rV) and recombinant fowlpox (rF) virusescontaining murine B7-1, ICAM-1, and LFA-3 genes as well as the human CEAgene (rV/F-CEA/TRICOM) have been previously described (Hodge et al.,Cancer Res 1999 Nov. 15; 59(22):5800-7; and Grosenbach et al., CancerRes 2001 Jun. 1; 61(11):4497-505). The murine GM-CSF-expressing rF virus(rFGM-CSF) has been previously described (Kass et al., Cancer Res 2001Jan. 1; 61(1):206-14). A recombinant Saccharomyces cerevisiae constructexpressing the full-length CEA protein (yeast-CEA) has been previouslydescribed (Bernstein et al., Vaccine 2008 Jan. 24; 26(4):509-21).Yeast-CEA was produced and heat-killed for these studies as previouslydescribed (Haller et al., Vaccine 2007 Feb. 9; 25(8):1452-63).

Vaccination Schedules

For serum cytokine analysis, CEA-Tg mice (n=2) were vaccinated with1×10⁸ pfu rVCEA/TRICOM or 4 YU/animal (1 YU=10⁷ yeast particles) ofyeast-CEA as previously described (Wansley et al., Clin Cancer Res 2008Jul. 1; 14(13):4316-25). For all other studies, the rV/F-CEA/TRICOMvaccine group, CEA-Tg mice were primed with 1×10⁸ pfu rV-CEA/TRICOMadmixed with 1×10⁷ pfu rF-GM-CSF on day 0, and boosted every 7 days with1×10⁸ pfu rF-CEA/TRICOM admixed with 1×10⁷ pfu rF-GM-CSF. For theremainder of the Examples and elsewhere according to this invention,this vaccine protocol will be generally designated as “rV/F-CEA/TRICOM”.In the yeast-CEA vaccine group, CEA-Tg mice were vaccinated every 7 dayswith yeast-CEA (4 YU/mouse). Mice receiving the combination ofrV/F-CEA/TRICOM and yeast-CEA vaccines were primed with 1×10⁸ pfurV-CEA/TRICOM, administered subcutaneously on the dorsal right flank,and with 4 YU/mouse of yeast-CEA, delivered subcutaneously on the innerlegs and shoulder blades. The separation of the yeast-CEA dose overmultiple sites has previously been described (Wansley, 2008, supra), andhas been employed here to separate not only the yeast-CEA vaccine, butalso the rV/F-CEA/TRICOM to target multiple draining lymph nodes in themouse. Mice in the combination group were boosted at 1-week intervalsfor the remainder of the study with 1×10⁸ pfu rF-CEA/TRICOM andyeast-CEA (4 YU/mouse).

Cytokine Expression Profiles

For serum cytokine analysis, vaccinated mice (see vaccination scheduleabove) were bled on days 0, 2, and 4 post-vaccination and serum wasisolated. Cytokine expression was analyzed using a Th1/Th2 andproinflammatory cytokine panel by Linco Diagnostic Services (St.Charles, Mo.). To measure cytokines secreted by CD8+ T-cells from micevaccinated with rV/F-CEA/TRICOM or yeast-CEA (n=5), CD8+ T-cells werebulk cultured and restimulated in the presence of CEA-572-579 peptide(GIQNSVSA, designated CEA-572 and represented herein by SEQ ID NO:11) orCEA-526-533 peptide (EAQNTTYL, designated CEA-526 and represented hereinby SEQ ID NO:12) (10 μg/ml) as previously described (Wansley et al.,2008, supra). Cytokine levels were measured using the mouse InflammatoryCytokine Cytometric Bead Array Kit and the mouse Th1/Th2 CytokineCytometric Bead Array kit (BD Biosciences, San Jose, Calif.) accordingto the manufacturer's instructions.

T-Cell Receptor (TCR) Profiles

RNA was isolated using the RNeasy Mini Kit (Qiagen, Inc., Valencia,Calif.) according to the manufacturer's instructions. RNA was then usedin RT-PCR reactions using the Invitrogen SUPERSCRIPT® First-StrandSynthesis System for RT-PCR (Invitrogen, 8 Carlsbad, Calif.) accordingto the manufacturer's instructions. Vα and Vβ genes were amplified usingprimers and conditions previously described for 19 Vα and 24 Vβ genes(Pannetier et al., Proc Natl Acad Sci USA 1993 May 1; 90(9):4319-23;Arden et al., Nature 1985 Aug. 29-Sep. 4; 316(6031):783-7.). PCRproducts were analyzed using the Agilent 2100 Bioanalyzer and AgilentDNA 1000 Reagent Kit (Agilent Technologies, Santa Clara, Calif.) byon-chip electrophoresis according to the manufacturer's instructions.Agilent 2100 Expert Software (version B.02.0651418 [Patch 01]) was usedto identify PCR products by size (bp) and quantity (nmol/L). For eachsample, quantities of each gene present were summed, and for each gene,a percent of the total TCR Vα or Vβ repertoire was calculated.

A mouse Vβ TCR screening panel (BD Pharmingen, San Jose, Calif.)consisting of monoclonal antibodies specific for mouse TCR Vβ 2, 3, 4,5.1 and 5.2, 6, 7, 8.1 and 8.2, 8.3, 9, 10, 11, 12, 13, 14, and 17 wereused to identify TCR Vβ expression at the protein level by flowcytometry using a FACScan cytometer (Becton Dickinson).

cDNA Oligoarray

CEA-Tg mice were either untreated or vaccinated 3 times at 1-weekintervals with rV/F-CEA/TRICOM or yeast-CEA. On day 33, splenocytes wereharvested and RNA was isolated. T- and B-cell activation, chemokines andchemokine receptors, and common cytokines cDNA oligoarrays(SABiosciences, Frederick, Md.) were used to investigate changes in geneexpression. Genes were considered up-regulated or down-regulated iftheir normalized intensity ratio was >2 or <0.5 (a 2-fold cutoff),respectively, according to manufacturer's recommendations.

CEA-Specific CTL Cell Lines and In Vitro Assays

CEA-526-specific and CEA-572-specific T-cell lines generated from micevaccinated with rV/F-CEA/TRICOM or yeast-CEA were maintained in culturewith CEA-526 or CEA-572 peptide (1 μg/ml) and IL-2 (10 U/ml) with freshirradiated APCs. To measure the ability of the T-cell lines to lyse¹¹¹In-labeled targets, various ratios of T cells were incubated withlabeled targets in triplicate at 37° C. and 5% CO₂ in 96-well U-bottomplates. In certain studies, anti-MHC class I blocking antibody (H2D^(b),BD Pharmingen) was used to distinguish between TCR-mediated and NK-likecytotoxicities. Radioactivity in supernatants was measured using agammacounter (Corba Autogamma, Packard Instruments, Downers Grove,Ill.). Percentage of tumor lysis was calculated as follows: % tumorlysis=[(experimental cpm−spontaneous cpm)/(maximum cpm−spontaneouscpm)]×100. To evaluate the avidity of CEA-specific CTL lines,tumor-killing activity was tested as previously described (Hodge et al,2005, J Immunol 174(10):5994-6004). Data were averaged and graphed as A% specific lysis. To normalize groups within each experiment, data werealso expressed as percentage of maximum lysis versus peptideconcentration. Finally, the natural logarithm of the normalized data wasplotted against peptide concentration. The avidity of each T-cellpopulation was defined as the negative log of the peptide concentrationthat resulted in 50% maximal target lysis (Hodge et al., 2005, supra andDerby et al, 2001, J Immunol 166(3):1690-1697) and was expressed in nM.The HIV-gag-390-398 peptide (SQVTNPANI, designated HIV-gag peptide andrepresented herein by SEQ ID NO:13 was used as a negative control inthis experiment. MHC class I-peptide tetramers specific for CEA-526 andCEA-572 were obtained from Beckman Coulter (Fullerton, Calif.). Whereindicated, CTL activity was converted to lytic units (LU), as describedby Wunderlich et al. (1994, “Induction and measurement of cytotoxic Tlymphocyte activity.” In: Coligan J, Kruisbeek A, Margulies D, ShevachE, Strober W (eds) Current Protocols in Immunology, Wiley, Hoboken,N.J.).

Tumor Therapy Studies

For therapy studies involving LL2-CEA tumors, 6- to 8-week-old femaleCEA-Tg mice were injected i.v. in the tail with 3×10⁵ LL2-CEA cells in avolume of 100 μl. Four days post-tumor implantation, mice were primedand then boosted as described above. To enumerate lung metastases, lungsfrom sacrificed mice were inflated, stained with India ink, and fixed inFekete's solution (Wexler, J Natl Cancer Inst 1966 April; 36(4):641-5).

Statistical Analysis

GraphPad Prism version 4.0a for Macintosh (GraphPad Software, San Diego,Calif.) was used to perform statistical analyses on in vivo data. A2-tailed, nonparametric Mann-Whitney test was performed for the averagenumber of tumors per mouse at day 45. A log-rank (Mantel-Cox) test wasperformed for mice bearing >10 pulmonary tumor nodules at day 45 whichwere deemed to have <1 week to live. All values were calculated at a 95%confidence interval and a p value <0.05 was considered significant.

Example 1

The following example shows the role of the immunotherapy vector ininducing cytokine and chemokine host innate immune responses that maysubsequently influence CEA-specific T-cell responses.

In this experiment, the role of the immunotherapy vector in inducingcytokine and chemokine host innate immune responses that maysubsequently influence CEA-specific T-cell responses was investigated.Briefly, CEA-Tg mice (n=2) were vaccinated with rV-CEA/TRICOM oryeast-CEA. Serum was collected at 0, 2, and 4 days, pooled and analyzedfor a panel of cytokines using a Th1/Th2 and pro-inflammatory cytokinepanel. As shown in FIG. 1, rV-CEA/TRICOM (closed squares) induces aTh1-type cytokine profile, where MIP1α, RANTES, GM-CSF, and IL-12p70levels are high and IL-5 levels are low (FIGS. 1A, B, C, and E,respectively). In contrast, yeast-CEA vaccination induces a mixedTh1/Th2 cytokine profile with increased levels of IL-6 (FIG. 1D), lowlevels of MIP1α, RANTES, IL-13, and IL-5 (FIGS. 1A, B, F, and I,respectively). Data are presented as pg/ml of cytokine on each day.These data show that vaccination with rV-CEA/TRICOM vs. yeast-CEAinduces expression of different cytokines, indicating that differentT-cell populations are induced by each of the vaccine platforms.

Example 2

The following example demonstrates that vaccination with rV/F-CEA/TRICOMvs. yeast-CEA induces distinct TCR repertoires.

This experiment sought to determine whether vaccination with eitherplatform induces CD8+ T-cell populations with distinct TCR repertoires.CEA-Tg mice (n=5 per group) were vaccinated with either rV/F-CEA/TRICOMor yeast-CEA as described in the Materials and Methods section above.Untreated mice served as a negative control (FIGS. 2A and 2D). Spleensfrom vaccinated mice were harvested 14 days post vaccination and pooled.RT-PCR reactions were performed using 19 Vα-specific and 24 Vβ-specificprimers. PCR products were then analyzed and the percentage of the totalTCR repertoire was calculated for each gene (FIG. 2; Astericks indicategenes that are uniquely expressed in T-cells from mice vaccinated withone vaccine compared to the other). The TCR Vα profiles of splenocytesfrom untreated mice and mice vaccinated with rV/F-CEA/TRICOM oryeast-CEA indicate that each group has a distinct TCR Vα expressionprofile (FIGS. 2A to C). The expression of 12 of the 19 Vα genes wassimilar between T-cells induced by both vaccines, while 7 Vα genes areunique to T-cell populations from one vaccine compared to the other(FIGS. 2B and C). Comparison of the Vβ repertoires from these sameanimals indicated that, with a few exceptions, the Vβ profiles alsodiffer among the two groups of mice (FIGS. 2D to E). The expression of14 of the 24 Vβ genes was similar between T-cells induced by bothvaccines, yet the vaccines induce unique Vβ genes as well. As shown inFIGS. 2E and F, 10 Vβ genes were uniquely expressed by T-cells fromeither rV/F-CEA/TRICOM or yeast-CEA-vaccinated CEA-Tg mice (V131, VO4,Vβ5.1, Vβ5.2, Vβ5.3, Vβ8.1, Vβ8.3, V139, Vβ10, and Vβ20). These dataindicate that the Vα and Vβ TCR repertoires of T-cells from untreatedmice and mice vaccinated with rV/F-CEA/TRICOM or yeast-CEA have bothshared and unique patterns of TCR gene expression. It is unknown,however, if these differences are due to different processing andpresentation of the CEA antigen by the different vector-infected cells,or to the vectors themselves. The TCR repertoires of T-cell linesspecific for two different CEA epitopes created from CEA-Tg micevaccinated with the two vector platforms are described in the examplesbelow.

Example 3

The following example shows that vaccination with rV/F-CEA/TRICOM oryeast-CEA induces both shared and unique gene expression in response tovector and antigen.

To investigate the effects of both vector and antigen on the geneexpression of splenocytes, cDNA oligoarrays were used to furthercharacterize the T-cell populations induced by vaccination withrV/F-CEA/TRICOM or yeast-CEA. The expression of 252 genes involved in T-and B-cell activation, chemokines, chemokine receptors, and cytokines bysplenocytes of CEA-Tg mice vaccinated with rV/FCEA/TRICOM or yeast-CEAwas investigated. Table 1 shows that for each array, both rV/FCEA/TRICOMand yeast-CEA induce changes in expression of the same genes, includingup-regulation of 26 genes by at least 2-fold, the majority of which areinvolved in cytokine signaling. In addition, both vaccines up-regulatedLtb4r2, a leukotriene receptor involved in chemotaxis of immune cells,genes involved in T-cell proliferation, such as secretedphosphoprotein-1 (Spp1, or osteopontin), and the tumor suppressor Inha.At the same time, each vaccine platform induces unique changes inexpression of several genes (Table 1, bold). Yeast-CEA down-regulatesgenes involved in chemotaxis of immune cells such as Cc112, Cxcl9, Ccr9,while rV/F-CEA/TRICOM does not alter the expression of any of the thesegenes. The results from this experiment indicate that the two vaccineplatforms induce changes in gene expression that are both shared andunique.

TABLE 1 Genes Involved in T-cell Chemokines and Vaccination and B-cellActivation Chemokine Receptors Cytokines rV/F-CEA/TRICOM/No treatmentGenes up- H60, Igbp1b, Il11, Il4 Inha (3.03), Ltb4r2, Il17c, Il17f,Inhba regulated >2-fold Bmp10, Bmp5 Fgf10, Gdf2, Gdf5, Gdf8, Ifna2,Ifna4, IfnbI, Il13, Il17b, Il25, Il19, Ilf10, Ilf5, Ilf6, Ilf8, Il20,Il3, Il9 Genes down- Ms4al, Sppl (4.00) Il1rn (4.29) regulated >2-foldYeast-CEA/No treatment Genes up- Rag1 (3.48) Bdnf, Ccl20, Cmtm2a, GdXregulated >2-fold H60 (3.25), Igbp1b (3.25), Cmtm5, Cxcl15, Gdf5, Fgf10,Gdf2, Gdf5, Gdf8, Il11 (3.25), Il4 Ccl17 Ifna2, Ifna4, IfnbI, Il13, Inha(4.92), Ltb4r2 (4.92), Il17b, Il25, Il19, Ilf10, Ilf5, Bmp10, Bmp5 Ilf6,Ilf8, Il20, Il3, Il9 Genes down- Tnfrsf13c (6.06) Ccl12 (3.03), Cc18(3.73) Gdf3(4.0), Il10, Csf-2, regulated >2-fold Ms4al (3.25), Spp1Ccr9, Ccrl2, Csf2, Cx3crl, Fasl, Tnf, Tnfrsf11b, Cxcl10, Cxcl13, Hif1a,Tnfsfl5 Inhbb, Lif (3.48), Gusb, Cxcl9 (4.92) Genes in bold areup-regulated/down-regulated specifically by splenocytes from micevaccinated with either rV/F-CEA/TRICOM or yeast-CEA, but not by both.Fold changes >3-fold are noted in parentheses

Example 4

The following example demonstrates that rV/F-CEA/TRICOM and yeast-CEAinduce functionally distinct T-cell populations.

To determine the antigen-specific response of T-cell populations inducedby the vaccines, the cytokines produced by T-cells from vaccinatedanimals after in vitro stimulation with either of two discrete CEAepitopes (CEA-572 and CEA-526) were investigated. FIG. 3A illustratesCEA protein showing the discrete, non-overlapping CEA-526 and CEA-572epitopes on the A3 loop of domain III.

Referring to FIG. 3, CEA-Tg mice (n=5) were primed with rV-CEA/TRICOM(solid bars) on day 0 and boosted on days 7 and 14 with rFCEA/TRICOM;CEA-Tg mice (n=5) were primed on day 0 and boosted on days 7 and 14 withyeast-CEA (open bars). On day 33, mice were sacrificed and spleenspooled and put into bulk cultures with either (FIG. 3B) CEA-526 or (FIG.3C) CEA-572 peptide for 7 days. IL-2, IL-10, TNF-α, IFN-γ, IL-5 and IL-4were measured by cytokine bead array (pg/ml/L×10⁶ cells) afterlymphocytes were restimulated for 24 hours with CEA-specific peptide orVSVN peptide control. All data have been normalized to the VSVN peptidecontrol.

It was observed that the two different CEA epitopes induce differentlevels of cytokine production from T cells from vaccinated animals.Higher levels of TNF-α are secreted in response to CEA-526 afterrV/F-CEA/TRICOM vaccination compared to yeast-CEA (FIG. 3B, closed bar),yet yeast-CEA vaccination produces significantly higher levels of TNF-αwhen T cells are stimulated with CEA-572 peptide (FIG. 3C, open bar).Also, T cells from yeast-CEA vaccination induce higher levels of IL-2compared to T cells from rV/F-CEA/TRICOM vaccination, when stimulatedwith the CEA-526 and CEA-572 peptides (FIGS. 3B and 3C, open bars).Similarly, after vaccination with rV/F-CEA/TRICOM, T cells induce higherlevels of IFN-γ compared to yeast-CEA vaccination in response to theCEA-526 and CEA-572 peptides (FIGS. 3B and 3C, closed bars).

The data also show that T-cells from vaccinated animals secretedifferent levels of various cytokines in response to a single CEAepitope. T-cells from mice vaccinated with yeast-CEA secrete IL-4,IL-10, TNF-α, IFN-γ, IL-5, and IL-2 in response to the CEA-572 epitope(FIG. 3C, open bars). On the other hand, T-cells from mice vaccinatedwith rV/F-CEA/TRICOM secrete significantly higher levels of IFN-γcompared to yeast-CEA in response to the CEA-572 peptide and lowerlevels of IL-10 and TNF-α in response to the CEA-572 epitope (FIG. 3C,closed bars). These results indicate that the T-cell populations inducedby vaccination with rV/F-CEA/TRICOM or yeast-CEA are antigen-specificand functionally distinct.

Example 5

The following example demonstrates that T-cell lines developed from micevaccinated with rV/F-CEA/TRICOM versus yeast-CEA have 14 distinct TCRrepertoires and functional avidity.

To further explore potential differences in the functionality of T cellsfrom mice vaccinated with either rV/F-CEA/TRICOM or yeast-CEA, T-celllines specific for either CEA-526 or CEA-572 peptide were created fromvaccinated CEA-Tg mice as described in the Materials and Methods.Briefly, CEA-Tg mice (n=5 per group) vaccinated with rV/F-CEA/TRICOM oryeast-CEA as described above. Two weeks after the final vaccination,spleens were harvested and pooled, and splenocytes were bulk culturedwith CEA-526 or CEA-572 peptide for 7 days. Lymphocytes wererestimulated with fresh peptide, IL-2, and irradiated APCs every 7 daysand kept in culture for in vitro experiments. TCR profile analysis wasconducted after 18 stimulation cycles.

Vα TCR profiles from the 4 cell lines indicate that the T-cellpopulations have shared and distinct Vα TCR repertoires. Referring toFIG. 4, Vα TCR repertoires of rV/F-CEA/TRICOM T-cell lines (black bars)maintained in the presence of (FIG. 4A) CEA-526 peptide and (FIG. 4B)CEA-572 peptide are shown. FIG. 4 also shows Vα TCR repertoires ofyeast-CEA T cell lines (white bars) maintained in the presence of (FIG.4C) CEA-526 peptide and (FIG. 4D) CEA-572 peptide. Results are expressedas percentage of total Vα chain TCR repertoire. Astericks indicate 31genes that are uniquely expressed in T cells from mice vaccinated withone vaccine compared to the other.

In T-cells from mice vaccinated stimulated with the CEA-526 epitope, theT-cells have shared expression of 16 of the 19 Vα genes and uniqueexpression of 3 Vα genes (FIGS. 4A and C). In T-cells from micevaccinated stimulated with the CEA-572 epitope, the T cells have sharedexpression of 15 of the 19 Vα genes and unique expression of 4 Vα genes(FIGS. 4B and D). Similar results were seen when Vβ TCR profiles wereanalyzed (data not shown). In addition, expression of selected Vβ TCRgenes of the T-cell lines were confirmed by flow cytometry usingcommercially available monoclonal antibodies (data not shown). Thesedata provide further evidence that the T-cell populations from micevaccinated with either rV/F-CEA/TRICOM or yeast-CEA are both vector- andantigen-specific.

To characterize the functional differences between T-cells from eithervector, the CEA-specific cytolytic activity of T-cells generated fromrV/FCEA/TRICOM was compared to that generated from yeast-CEAvaccination. The purity of the T-cell line cultures was confirmed viacell surface staining with monoclonal antibodies to identify CD8, CD4,and NK cells followed by flow cytometry (data not shown). In addition,tetramer staining using MHC class I-peptide tetramers specific forCEA-526 or CEA-572 confirmed peptide specificity for the T-cell lines(data not shown).

Briefly, T cell lines generated from rV/F-CEA/TRICOM vaccination andspecific for (FIG. 5A) CEA-526 peptide and (FIG. 5C) CEA-572 peptidewere incubated with peptide-pulsed ¹¹¹In-labeled EL-4 cell targets atthe indicated ratios for 4 h. T-cell lines generated from yeast-CEAvaccination and specific for (FIG. 5B) CEA-526 peptide and (FIG. 5D)CEA-572 peptide were also incubated with ¹¹¹In-labeled EL-4 cell targetsat the indicated ratios for 4 h. Referring to FIG. 5, EL-4 cells pulsedwith CEA-572 and CEA-526 peptides are represented by solid squaresconnected by a solid line, and ¹¹¹In-labeled EL-4 cells pulsed withVSVNP (negative control) are represented by open circles connected by adotted line. To determine T-cell avidity, (FIG. 5B, inset)CEA-526-specific T-cell lines from rV/F-CEA/TRICOM (closed squares) andyeast-CEA (open circles) were incubated with ¹¹¹In-labeled EL-4 cells inthe presence of various concentrations of CEA-526 (or HIV-gag control)peptide ranging from 1 μM to 0 μM for 4 h. T-cell lines specific forCEA-572 epitope, generated from mice vaccinated with rV/F-CEA/TRICOM(FIG. 5E) or yeast-CEA (FIG. 5F), were also used in cytolytic T-cellassays with ¹¹¹In-labeled LL2-CEA and normalized to LL2 (negativecontrol) tumor targets at various ratios. Bars indicate standard errorfrom triplicate wells.

FIGS. 5A and 5B show that a CEA-526 peptide-specific T-cell linegenerated from rV/F-CEA/TRICOM has higher lytic activity compared to aT-cell line generated from yeast-CEA vaccination. T-cell lines specificfor the CEA-572 epitope both demonstrated similar levels of cytolyticactivity (FIGS. 5C and 5D). FIG. 5B inset shows that theCEA-526-specific T-cell line generated from rV/F-CEA/TRICOM vaccinationhad a 23.3-fold higher avidity than the CEA-526-specific T-cell linegenerated from yeast-CEA vaccination.

The CEA-572-specific T-cell lines were also used in a CTL assaytargeting ¹¹¹In-labeled LL2-CEA cells. CEA-572-specific T cells frommice vaccinated with either rV/F-CEA/TRICOM or yeast-CEA were culturedfor 20 weeks prior to this assay. Both T-cell lines lyse LL2-CEA targetsand lysis decreases as the ratio of T-cells to effector cells (LL2-CEAtargets) decreases (FIGS. 5E and 5F). These results indicate that bothT-cell lines are capable of lysing CEA-expressing cells, although theT-cell line from mice vaccinated with yeast-CEA (FIG. 5F) had a higherlevel of activity compared to the T-cell line from mice vaccinated withrV/FCEA/TRICOM (FIG. 5E) when LL2-CEA cell lysis was normalized to thatof LL2 cells.

To confirm that the cell lysis observed in FIGS. 5E and 5F wasTCR-mediated and not due to NK cell activity, CTL experiments withblocking monoclonal antibodies specific for MHC class I molecules wereperformed with LL2-CEA tumor targets and normalized to LL2 target cellsas a control, and showed that the presence of MHC class I blockingantibody abrogated cell lysis. The lack of NK cell-mediated lysis wasfurther confirmed in a CTL using YAK1 targets, which found that thepresence of MHC class blocking antibody abrogated YAK1 cell lysis by thevarious T-cell lines. Together, these results indicate that the lyticactivity of T-cell lines created from different vectors targeting thesame CEA-epitope is TCR-mediated and levels of cell lysis are similarwhen targeting peptide-pulsed target cells, although their ability tolyse CEA-expressing tumor targets differs. Additionally, the avidity ofrV/F-CEA/TRICOM-induced T-cell lines may be higher than that of T-celllines created from yeast-CEA vaccination. These results furthercharacterize the T-cell populations from mice vaccinated withrV-CEA/TRICOM or yeast-CEA as platform-specific.

Example 6

The following example demonstrates that combining rV/F-CEA/TRICOM andyeast-CEA is an efficacious antitumor therapy in a murine orthotopicpulmonary metastasis model.

Studies were conducted to determine if concurrent administration of thetwo vaccines would generate antitumor activity superior to vaccinationwith either vaccine platform alone. Briefly, CEA-Tg mice were injectedi.v. with LL2-CEA tumor cells. On day 4, mice were primed withrV/F-CEA/TRICOM (n=10), yeast-CEA (n=14), or rV/F-CEA/TRICOM andyeast-CEA (n=10); a control group (n=17) received no treatment. Micewere boosted every 7 days for the duration of the experiment. TherV/F-CEA/TRICOM group was boosted with rV/F-CEA/TRICOM. The yeast-CEAgroup was boosted with yeast-CEA only. The combination group was boostedwith rV/F-CEA/TRICOM and yeast-CEA. For these studies, rV/F-CEA/TRICOMwas injected s.c. on the dorsal right flank while 1 YU yeast-CEA wasdelivered s.c. to each inner leg and shoulder blade to target multipledraining lymph nodes. On day 45, mice were sacrificed and lungs wereharvested, stained, and fixed. The data shown in FIG. 6 represent thenumber of lung metastases per mouse from two separate experiments(indicated by open vs. closed symbols). The bar indicates the averagenumber of metastases per mouse p=0.015 comparing untreated mice with therV/F-CEA/TRICOM and yeast-CEA combination group.

Untreated mice had an average of 10.84 tumors per mouse (+2.41). Micevaccinated with rV/FCEA/TRICOM had an average of 7.50 metastases permouse (+2.02), and mice vaccinated with yeast-CEA had an average of 9.71metastases per mouse (+1.22). However, mice vaccinated with thecombination of rV/F-CEA/TRICOM and yeast-CEA had 2.80 metastases permouse (+0.77); this combination group was the only group with asignificantly lower number of metastases compared to the untreatedcontrol (p=0.015). Also, the maximum number of metastases per mouse forthe untreated, rV/F-CEA/TRICOM, and yeast-CEA groups was 36, 24, and 18,respectively, while the maximum number of metastases in the combinationgroup was 7. Moreover, the log-rank test (mice bearing >9 pulmonarytumor nodules on day 45; assumed to have <1 week to live) showedstatistical significance between untreated mice and the mice thatreceived the combination of rV/F-CEA/TRICOM and yeast-CEA (p=0.0027).Also, there was statistical significance between mice treated withrV/F-CEA/TRICOM alone versus concurrent vaccination with rV/F-CEA/TRICOMand yeast-CEA (p=0.0293). In addition, there was statisticalsignificance between mice treated with yeast-CEA alone versus concurrentvaccination with rV/F-CEA/TRICOM and yeast-CEA (p=0.0017). Theseresults, taken together, indicate that concurrent administration ofrV/F-CEA/TRICOM and yeast-CEA vaccines can increase antitumor efficacy.

As discussed above, published reports comparing vaccine platforms havehistorically concluded that one is more effective than the other atstimulating the immune system, and have thus recommended furtherdevelopment of the more effective platform for clinical studies(Riezebos-Brilman et al., Gene Ther 2007 December; 14(24):1695-704; andCasimiro et al., J Virol 2003 June; 77(11):6305-13). The resultsprovided herein have demonstrated that the T-cell population elicited byeach platform displayed unique and shared phenotypic and functionalresponses to different CEA epitopes. The results presented herein showfor the first time that (a) 2 vaccine platforms targeting the sameantigen induce distinct T-cell populations, (b) induction of theseT-cell populations is both vector- and antigen-specific, and (c) thevaccines can be used concurrently in an antitumor model to improveantitumor efficacy.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

1-78. (canceled)
 79. A method to prevent or treat at least one symptomof a disease or condition in an individual, comprising administering twoimmunotherapy compositions within a dosing period, the two immunotherapycompositions comprising: a) a first immunotherapy composition comprisinga recombinant Ad5 adenovirus comprising a nucleic acid sequence encodingone or more peptides; and b) a second immunotherapy compositioncomprising one or more peptides and either a whole inactivated yeast oryeast lysate; wherein the one or more peptides of the first and secondimmunotherapy compositions are derived from the same one or moreproteins.
 80. The method of claim 79, wherein the whole inactivatedyeast is a whole, heat-killed yeast.
 81. The method of claim 79, whereinthe whole inactivated yeast is from Saccharomyces.
 82. The method ofclaim 79, wherein the first and second immunotherapy compositions areadministered to different sites in the individual.
 83. The method ofclaim 79, wherein the first and second immunotherapy compositions areadministered to the same site or to adjacent sites in the individual.84. The method of claim 79, further comprising boosting the individualwith one or both of the immunotherapy compositions.
 85. The method ofclaim 84, wherein boosting the individual is with both immunotherapycompositions.
 86. The method of claim 85, further comprising boostingthe individual with a third immunotherapy composition comprising arecombinant virus comprising the virus genome or portions thereof thatis different from the first immunotherapy composition.
 87. The method ofclaim 79, wherein the individual is further treated with chemotherapyand/or with radiation therapy.
 88. The method of claim 79, wherein thedosing period comprises no more than 2 days.
 89. The method of claim 88,wherein the dosing period comprises no more than 1 day.
 90. The methodof claim 89, wherein the dosing period comprises no more than 12 hours.91. The method of claim 90, wherein the dosing period comprises no morethan 8 hours.
 92. The method of claim 91, wherein the dosing periodcomprises no more than 4 hours.
 93. The method of claim 92, wherein thedosing period comprises no more than 3 hours.
 94. The method of claim93, wherein the dosing period comprises no more than 2 hours.
 95. Themethod of claim 94, wherein the dosing period comprises no more than 1hour.
 96. The method of claim 95, wherein the dosing period comprises nomore than 1, 2, 3, 4, 6, 7, 8, 9, or 10 minutes.
 97. The method of claim79, wherein administering within the dosing period further comprisesadministering the first and second immunotherapy compositionssimultaneously.
 98. The method of claim 79, wherein administering withinthe dosing period further comprises administering the first and secondimmunotherapy compositions sequentially.
 99. A method to induce atherapeutic immune response against one or more proteins in anindividual, comprising administering two immunotherapy compositionswithin a dosing period, the two immunotherapy compositions comprising:a) a first immunotherapy composition comprising a recombinant Ad5adenovirus comprising a nucleic acid sequence encoding at least onepeptide; and b) a second immunotherapy composition comprising at leastone peptide and either a whole inactivated yeast or yeast lysate;wherein the peptides of the first and second immunotherapy compositionsare derived from the same one or more proteins.
 100. The method of claim99, wherein the whole inactivated yeast is a whole, heat-killed yeast.101. The method of claim 99, wherein the whole inactivated yeast is fromSaccharomyces.
 102. The method of claim 99, wherein the first and secondimmunotherapy compositions are administered to different sites in theindividual.
 103. The method of claim 99, wherein the first and secondimmunotherapy compositions are administered to the same site or toadjacent sites in the individual.
 104. The method of claim 99, furthercomprising boosting the individual with one or both of the immunotherapycompositions.
 105. The method of claim 104, wherein boosting theindividual is with both immunotherapy compositions.
 106. The method ofclaim 105, further comprising boosting the individual with a thirdimmunotherapy composition comprising a recombinant virus comprising thevirus genome or portions thereof that is different from the firstimmunotherapy composition.
 107. The method of claim 99, wherein theindividual is further treated with chemotherapy and/or with radiationtherapy.
 108. The method of claim 99, wherein the dosing periodcomprises no more than 2 days.
 109. The method of claim 108, wherein thedosing period comprises no more than 1 day.
 110. The method of claim109, wherein the dosing period comprises no more than 12 hours.
 111. Themethod of claim 110, wherein the dosing period comprises no more than 8hours.
 112. The method of claim 111, wherein the dosing period comprisesno more than 4 hours.
 113. The method of claim 112, wherein the dosingperiod comprises no more than 3 hours.
 114. The method of claim 113,wherein the dosing period comprises no more than 2 hours.
 115. Themethod of claim 114, wherein the dosing period comprises no more than 1hour.
 116. The method of claim 115, wherein the dosing period comprisesno more than 1, 2, 3, 4, 6, 7, 8, 9, or 10 minutes.
 117. The method ofclaim 99, wherein administering within the dosing period furthercomprises administering the first and second immunotherapy compositionssimultaneously.
 118. The method of claim 99, wherein administeringwithin the dosing period further comprises administering the first andsecond immunotherapy compositions sequentially.